Methods and compositions for derepression of IAP-inhibited caspase

ABSTRACT

The invention provides isolated agents having novel chemical structures and possessing superior activity as derepressors of IAP inhibited caspase. The invention further provides a method of derepressing an IAP-inhibited caspase. The invention further provides assay methods employing labeled compounds of the invention, especially fluorescent labeled compounds.

This invention was made with government support under grant numberCA78040 awarded by The National Institute of Health/National CancerInstitute. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The present invention relates generally to molecular medicine and morespecifically to compositions and methods for altering molecularinteractions involved in regulating programmed cell death.

Normal tissues in the body are formed either by cells that have reacheda terminally differentiated state and no longer divide or by cells thatdie after a period of time and are replaced from a pool of dividingcells. For example, nervous tissue is formed early in development andthe cells of the nervous system reach a terminally differentiated statesoon after birth. In contrast, the body has a number of self renewingtissues such as skin, gut, bone marrow and sex organs which undergo abalanced flux of cell birth and death. This flux, which results in theproduction of 50-70 billion cells per day in an average adult andamounting to a mass of cells equivalent to an entire body weight over ayears time, is balanced by the regulated eradication of an equivalentnumber of cells. In self renewing tissues the eradication is maintained,in part, due to the process of programmed cell death, known asapoptosis, in which the cells are genetically “programmed” to die aftera certain period of time.

Apoptosis is particularly prominent during the development of anorganism, where cells that perform transitory functions are programmedto die after their function no longer is required. In addition,apoptosis can occur in cells that have undergone major geneticalterations, thus providing the organism with a means to rid itself ofdefective and potentially cancer forming cells. Apoptosis also can beinduced due to exposure of an organism to various external stimuli,including, for example, bacterial toxins, ethanol and ultravioletradiation. Chemotherapeutic agents for treating cancer also are potentinducers of apoptosis.

The regulation of programmed cell death is a complex process involvingnumerous pathways and, on occasion, defects occur in the regulation ofprogrammed cell death. Given the critical role of this process inmaintaining a steady-state number of cells in a tissue or in maintainingthe appropriate cells during development of an organism, defects inprogrammed cell death often are associated with pathologic conditions.It is estimated that either too little or too much cell death isinvolved in over half of the diseases for which adequate therapies donot currently exist.

Various disease states occur due to aberrant regulation of programmedcell death in an organism. For example, defects that result in adecreased level of apoptosis in a tissue as compared to the normal levelrequired to maintain the steady-state of the tissue can result in anincreased number of cells in the tissue. Such a mechanism of increasingcell numbers has been identified in various cancers, where the formationof a tumor occurs not because the cancer cells necessarily are dividingmore rapidly than their normal counterparts, but because the cells arenot dying at their normal rate.

Thus, a need exists for agents capable of modulating programmed celldeath pathways and methods for treating individuals experiencingdiseases associated with aberrant regulation of programmed cell death.The present invention satisfies this need and provides additionaladvantages as well.

SUMMARY OF THE INVENTION

The invention provides isolated agents having one of the structures TPI1577-1, TPI 1577-2, TPI 1577-3, TPI 1567-5, TPI 1577-6, TPI 1577-7, TPI1577-8, TPI 1577-9, TPI 1567-11, TPI 1567-12, TPI 1567-13, TPI 1567-14,TPI 1567-23, TPI 1567-24, TPI 1567-18, TPI 1572-8, TPI 1572-15, TPI1572-16, TPI 1572-10, TPI 1572-11; TPI 1572-14; TPI 1572-17, TPI1572-18, TPI 1572-19, TPI 1572-20, TPI 1572-21, TPI 1572-22 or TPI1572-23. These compounds are derepressors of IAP-inhibited caspase. Theinvention further provides a method of derepressing an IAP-inhibitedcaspase. The method comprises contacting an IAP-inhibited caspase withan effective amount of one of the agents. The invention also provide amethod for promoting apoptosis in a cell and for reducing the severityof a pathology characterized by reduced levels of apoptosis.

The invention further provides assay methods for identifying an IAPinhibited caspase derepressor. One method involves providing a labeledcandidate agent and measuring a label signal in the presence and absenceof IAP or a fragment of IAP. The difference in label signal in thepresence and absence of IAP or fragment thereof is a measure of thedegree of binding of the candidate agent to IAP or fragment thereof. Themethod optionally includes creating a binding curve, plotting theconcentration of either IAP (or its fragment) or the candidate agentagainst the difference between bound and unbound label signal.

The invention further provides another assay method for identifying anIAP inhibited caspase derepressor. A labeled candidate agent is firstprovided. A label signal is measured for the labeled candidate agent inthe absence of IAP and fragments thereof. Then a label signal isobtained for the labeled candidate agent in the presence of a knownIAP-binding agent and IAP or a fragment thereof. The difference betweenthe first and second label signals corresponds to the relative affinityof the candidate agent for IAP, and is thus predictive of the IAPinhibited caspase derepressor activity of the candidate agent. Themethod optionally includes creating a binding curve, plotting theconcentration of either IAP (or its fragment) or the candidate agentagainst the difference between bound and unbound label signal.

The invention provides isolated agents having a core peptide selectedfrom the group consisting of Core peptides 4 through 39 and 42 through55, wherein the agent derepresses an IAP-inhibited caspase. Alsoprovided is an isolated agent having a core structure selected from anyof the structures shown in FIGS. 5, 9, 10, 14B, 21-24, 34, 35 and 36,wherein the agent derepresses an IAP-inhibited caspase. The inventionfurther provides a method of derepressing an IAP-inhibited caspase. Themethod consists of contacting an IAP-inhibited caspase with an effectiveamount of an agent to derepress an IAP-inhibited caspase, the agenthaving a core motif selected from the group consisting of a core peptidehaving a sequence set forth in any of Core peptides 4 through 39 and 42through 55; a core structure selected from the group consisting ofTPI759, TPI 882, TPI 914 or TPI 927; and a core structure selected fromTPI 1391, TPI 1349, TPI 1396, TPI 1509, TPI 1540, TPI 1400, TPI 792 andTPI 1332. The invention also provides methods for promoting apoptosis ina cell and for reducing the severity of a pathology characterized byreduced levels of apoptosis. Methods for identifying agents thatderepress an IAP-inhibited caspase further are provided.

The invention further provides a homogeneous radioassay method ofidentifying an agent that binds IAP. The method includes providing ascintillation bead that is linked to IAP or a fragment of IAP and acompound known to bind to IAP or a fragment of IAP. The known IAPbinding compound is radiolabeled, for example with a tritium label. Thescintiallation bead is then contacted with the radiolabeled IAP bindingcompound in the presence of a candidate compound. Binding of the knownIAP binding compound is measured by scintillation counting by thescintillation proximity method. A decrease in scintillation counts inthe presence of a candidate compound indicates that the candidatecompound competes with the known IAP binding compound. Thus, a candidatecompound that causes a decrease in scintillation counts in this assay isidentified as an IAP binding compound. The binding constant of thecandidate compound can be prepared by titrating the candidate compoundagainst a known concentration of known IAP binding compound, forexample.

The invention also provides another method of identifying an agent thatbinds IAP. The method is a non-homogeneous competition assay, whichinvolves immobilizing the IAP or fragment of IAP on a support, such as acommercially available 96 well plate. The immobilized IAP or fragment ofIAP is then contacted with a known compound that binds IAP. The knowncompound is labeled with a suitable label, such as a fluorescent label,a radiolabel, biotin, an ezyme, etc. The known compound and the IAP orfragment of IAP form a bound complex, which remains immobilized evenupon washing. A signal can be obtained from the bound complex, whichrepresents a negative control. The bound complex is contacted with acandidate agent. If the candidate agent competitively binds IAP or afragment of IAP, it will displace the labeled known compound from thebound complex. After washing, it is a label signal is then determinedfor the complex. A decrease in the label signal from the negativecontrol label signal indicates that the candidate compound competitivelybinds IAP or a fragment of IAP. Thus, a candidate compound that causes adecrease in label signal is identified as an IAP binding compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of values obtained for the ratio of V_(max) (whereV_(max) is equal to RFU/min) for hydrolysis ofAcetyl-DEVD-7-amino-4-trifluoromethyl-coumarin (Ac-DEVD-AFC) in thepresence and absence of each species of the TPI 1328 library, composedof mixtures of hexapeptides.

FIG. 2 shows a table listing individual tetrapeptides of the TPI 1313library and the ratio of V_(max) for hydrolysis of Ac-DEVD-AFC in thepresence and absence of each peptide species. The ratio=(V_(max) whenpeptide, caspase 3 and XIAP are present)/(V_(max) when caspase 3 andXIAP are present).

FIG. 3 shows structures for the individual species of tetrapeptides inthe TPI 1313 library.

FIG. 4 shows structures of the defined functionalities in the mixturesfound to be derepressors of an XIAP-inhibited caspase in the TPI914N-acyltriamine positional scanning combinatorial library. The chemicalname listed below each box is the reagent from which the R group wasderived. Each functional group has the same stereochemistry as thereagent from which it was derived.

FIG. 5 shows structures for the individual compounds found to bederepressors of an XIAP-inhibited caspase in the TPI914 N-acyltriaminelibrary. The chemical name listed at each table entry is the reagentfrom which the R group was derived. Each functional group has the samestereochemistry as the reagent from which it was derived.

FIG. 6 shows structures of the defined functionalities in the mixturesfound to be derepressors of an XIAP-inhibited caspase in the TPI927polyphenylurea positional scanning combinatorial library. The chemicalname in each box is the reagent from which the R group was derived. Eachfunctional group has the same stereochemistry as the reagent from whichit was derived. For structures 25, 73, 86 and 88, where the corestructure of the molecule is modified, the resulting modified corestructure and R group is shown.

FIG. 7 shows structures of the defined functionalities in the mixturesfound to be derepressors of an XIAP-inhibited caspase in the TPI882C-6-acylamino bicyclic guanidine library. The chemical name in each boxis the reagent from which the R group was derived. Each functional grouphas the same stereochemistry as the reagent from which it was derived.

FIG. 8 shows structures of the defined functionalities in the mixturesfound to be derepressors of an XIAP-inhibited caspase in the TPI759N-benzyl-1,4,5-trisusbstituted-2,3-diketopiperazine positional scanningcombinatorial library. The chemical name listed below each box is thereagent from which the R group was derived. Each functional group hasthe same stereochemistry as the reagent from which it was derived.

FIG. 9 shows structures for individual compounds found to bederepressors of an XIAP-inhibited caspase in the TPI927 polyphenylurealibrary. The chemical name in each box is the reagent from which the Rgroup was derived. Each functional group has the same stereochemistry asthe reagent from which it was derived.

FIG. 10 shows structures for individual compounds found to bederepressors of an XIAP-inhibited caspase in the TPI882 C-6-acylaminobicyclic guanidine library. The chemical name in each box is the reagentfrom which the R group was derived. Each functional group has the samestereochemistry as the reagent from which it was derived.

FIG. 11 shows dose response of mixtures identified as derepressors ofXIAP-inhibited caspase from the TPI 1239 library. Values shown are forthe ratio of V_(max) for hydrolysis of AC-DEVD-AFC in the presence andabsence of each mixture. TPI 1239 mixtures were present at the doseslisted at the top of each column.

FIG. 12 shows the structures ofL-3-(2-thienyl)-alanyl-L-(2-naphthyl)-alanyl-L-p-chloro-phenylalanyl-L-(e-fluorenylmethyloxycarbonyl)-lysine(TPI792-33; Core peptide 16) and L-3-(2-thienyl)-alanyl,L-(2-naphthyl)-alanyl-D-(e-fluorenylmethyloxycarbonyl)-lysyl-L-(e-fluorenylmethyloxycarbonyl)-lysine(TPI792-35; Core peptide 17).

FIG. 13 shows the effects of VP-16 (etoposide), TPI792-35, TPI792-33 andthe SMAC AVPI tetrapeptide (SEQ ID NO:4) on prostate cancer cellviability.

FIG. 14 shows the generalized structures for phenyl urea compounds inthe TPI 1396 library and diketopiperazine compounds in the TPI 1391library (Panel A) and structures for compounds TPI 1391-28, TPI 1391-21,TPI 1396-34, TPI 1396-22, TPI 1396-11, TPI 1396-12 (Panel B).

FIG. 15 shows concentration-dependent killing of Jurkat leukemia cellsby TPI 1391-28 and TPI 1396-34.

FIG. 16 shows killing of Jurkat leukemia cells by TPI 1391-28 and TPI1396-34 compared to control compounds having similar corepharmacophores, respectively, but different R groups.

FIG. 17 shows a comparison of the effects of TPI 1396-34 and TPI 1391-28on normal bone marrow cells versus Jurkat leukemia cells.

FIG. 18 shows the effects of over-expression of wild-type XIAP on theapoptogenic activity of TPI 1396-34.

FIG. 19 shows the effects of over-expression of wild-type XIAP on theapoptogenic activity of TPI 1396-34.

FIG. 20 shows structures for TPI792-3, TPI792-9, TPI792-15, TPI792-17,TPI792-19, TPI792-22, TPI792-27, TPI792-33 and TPI792-35.

FIG. 21A shows structures for TPI 1349-1 through TPI 1349-34 along withrespective molecular weights, masses and lowest concentration of eachagent having a ratio of 1.8 or higher in SMAC competition assays. FIG.21B shows the activity of TPI 1349-1 through TPI 1349-34 in thederepression assay using full length XIAP. FIG. 21C shows the activityof TPI 1349-1, -3, -8, -13, -23, and -28 using both full length XIAP andXIAP BIR2 domain. FIG. 21D shows the activity of TPI 1349-1, -3, -8,-13, -23, and -28 using cIAP BIR2 domain.

FIG. 22A shows structures of TPI 1396-1 through TPI 1396-65 along withrespective molecular weights, masses and lowest concentration of eachagent having a ratio of 1.8 or higher in SMAC competition assays. FIG.22B shows the activity of TPI 1396-1 through TPI 1396-36 in thederepression assay using full length XIAP. FIG. 22C shows the activityof TPI 1396-37 through TPI 1396-65 in the derepression assay using filllength XIAP. FIG. 22D shows a table indicating the activities of TPI1396-11, -12, -22, -28, and -34 in the derepression assay using fulllength XIAP and the XIAP BIR2 domain. FIG. 22E shows the activity of TPI1396-11, -12, -22, -28, and -34 at 50 μg/ml using XIAP BIR2 domain. FIG.22F shows the activity of TPI 1396-11, -12, -22, -28, and -34 at 100μg/ml using full length XIAP and Caspase 3 or 7. FIG. 22G shows theactivity of TPI 1396-11, -12, -22, -28, and -34 at 100 μg/ml using cIAPBIR2 domain.

FIG. 23A shows structures of TPI 1391-1 through TPI 1391-36 along withrespective molecular weights, masses and lowest concentration of eachagent having a ratio of 1.8 or higher in SMAC competition assays. FIG.23B shows the activity of TPI 1391-1 through TPI 1391-36 at 100 μg/ml inthe derepression assay using full length XIAP. FIG. 23C shows theactivity of TPI 1391-1 through TPI 1391-36 at 25 μg/ml in thederepression assay using full length XIAP. FIG. 23D shows a tableindicating the activities of TPI 1391-1, -4, -5, 7, -17, -21, -25, -28,-34 and -35 in the derepression assay using full length XIAP. FIG. 23Eshows a comparison of the activities of TPI 1391-1, -4, -5, 7, -17, -21,-25, -28, -34 and -35 in the derepression assay using full length XIAPor XIAP BIR2 domain. FIG. 23F shows the activity of TPI 1391-1, -4, -5,7, -17, -21, -25, -28, -34 and -35 using cIAP BIR2 domain.

FIG. 24 shows structures of TPI 1400-1 through TPI 1400-58 along withrespective molecular weights, masses and lowest concentration of eachagent having a ratio of 1.8 or higher in SMAC competition assays. FIG.24B shows the activity of TPI 1400-1 through TPI 1400-28 at 25 μg/ml inthe derepression assay using full length XIAP. FIG. 24C shows theactivity of TPI 1400-1 through TPI 1400-28 at 10 μg/ml in thederepression assay using full length XIAP. FIG. 24D shows the activityof TPI 1400-29 through TPI 1400-58 at 25 μg/ml in the derepression assayusing full length XIAP. FIG. 24E shows the activity of TPI 1400-29through TPI 1400-58 at 10 μg/ml in the derepression assay using fulllength XIAP. FIG. 24F shows a table indicating the activities of TPI1400-6, -7, 13, -14, -33, -37, -43, -44 in the derepression assay usingfull length XIAP. FIG. 24G shows a comparison of the activities of TPI1400-6, -7, 13, -14, -33, -37, -43, -44 in the derepression assay usingfull length XIAP or XIAP BIR2 domain. FIG. 24H shows the activity of TPI1400-6, -7, 13, -14, -33, -37, -43, -44 using cIAP BIR2 domain.

FIGS. 25 a and b show screening of small molecule poly-phenylureacompounds in a Caspase derepression assay to identify compounds thatovercome XIAP-mediated repression of Caspase-3. FIG. 25 a shows theresults using aliquots from the poly-phenylurea library mixtures andFIG. 25 b shows the results for 36 individual compounds based ondeconvolution of the poly-phenylurea library.

FIGS. 26 a, b, c, d, e, and f show characterization of the biochemicalmechanism of poly-phenylurea compounds. FIGS. 26 a, b, c, and d show theresults of a Caspase derepression assay using poly-phenylurea compoundswith XIAP and Caspase-3 (a), XIAP and Caspase-9 (b), BIR-2 and Caspase-3(c) and p35 and Caspase-3 (d). FIG. 26 e shows a binding assay wherebiotinylated SMAC (7-mer) was adsorbed to Neutravidin-coated plates thenGST-XIAP was added with or without compounds. Bound GST-XIAP wasdetected with an anti-GST antibody. FIG. 26 f shows a binding assaywhere GST-XIAP was adsorbed to plates and then incubated withbiotinylated-SMAC (7-mer) with or without compounds. Boundbiotinylated-SMAC peptide was detecting by a streptavidin-europium-basedfluorescence method.

FIGS. 27 a, b, c, d, e, and f show characterization of cellular activityof poly-phenylurea compounds. FIGS. 27 a and b show cell death afterincubation of Jurkat leukemia cells with various poly-phenylureacompounds (a) or TPI 1396-34 (b). FIG. 27 c shows Caspase3/7 activityafter incubation of Jurkat cells with various compounds. FIG. 27 d showscell death in Jurkat cells cultured with various concentrations of TPI1396-34 with or without zVAD-fink. FIG. 27 e shows cell death afterincubation of U937 cells that stably over-express XIAP or neomycin withTPI 1396-34. Inset shows immunoblot analysis of lysates prepared fromthe U937 cells. FIG. 27 f shows cell death of HeLa cells transfectedwith XIAP, Bcl-XL or CrmA and incubated with TPI 1396-34. FIG. 27 gshows viability of control and SV40-transfected cells treated with TPI1396-34.

FIGS. 28 a, b, c and d show broad anti-tumor activity of TPI 1396-34.FIG. 28 a shows the result of cell growth of sixty human tumor celllines cultured with TPI 1396-34 or TPI 1396-28 compared to cells treatedwith solvent alone. Each line represents a tumor cell line. FIG. 28 bshows the effect of TPI 1396-34 on normal versus malignant cells. FIG.28 c shows the mean (+/−standard deviation) percent apoptosis of CLLB-cells from five patients cultured with various poly-phenylureacompounds. FIG. 28 d shows cell death of AML cells isolated from 5patients and cultured with various concentrations of TPI 1396-34. Allsamples were treated with active TPI 1396-34 and inactive TPI 1396-28 aswell as AVPI peptide, but the complete data set is shown only for AML-1.Comparable results were obtained with the other samples.

FIG. 29 shows the effects of TPI 1396-12, TPI 1396-22, and TPI 1396-11on 60 tumor cell lines from the NCI-60 cell panel on cell growthcompared to cells cultured with solvent diluent alone.

FIGS. 30 a and b show TPI 1396-34 sensitizes cancer cells tochemotherapy and TRAIL. FIG. 30 a shows viability of DU145 prostatecancer cells cultured with Etoposide (VP16), Doxorubicin (DOX) orPaclitaxel (Tax) with or without TPI 1396-34. FIG. 30 b shows viabilityof cancer cell lines treated with various concentrations of TRAIL aloneor in combination with TPI 1396-34.

FIGS. 31 a, b, c and d show the effect of combination of conventionalchemotherapeutic agents with TPI 1396-34 on various tumor cell lines.The viability of DUI45 (a), PPC1 (b), PC3 (c), and H460 (d) cellscultured with various concentrations of TPI 1396-34 and variousconcentrations of chemotherapeutic drugs is shown.

FIGS. 32 a, b and c show anti-tumor activity of TPI 1396-34 inclonogenic survival assays and tumor xenograft studies. FIG. 32 a showscolony number of two prostate cancer cell lines, PC-3 and LNCaP,cultured with TPI 1396-34. Control compound is represented by the bars,showing only the 10 μM dose results. FIG. 32 b shows tumor size inBalb/C nu−/nu− mice injected with PPC1 prostate cancer cells aftertreatment with TPI 1396-34. The inset shows tumor weight in micesacrificed at 24 days after compound injections. FIG. 32 c shows tumorvolume and tumor weight in Balb/C nu−/nu− mice injected with HCT116colon cancer cells. On days 6, 7, and 8 mice were treated with TPI1396-34 (I) or solvent control (C) and tumor volume was measured. On day19, the mice were sacrificed and the tumors were weighed. Bars representthe median tumor size or weight.

FIGS. 33 a and b show anti-tumor activity of poly-phenylurea compoundsin a tumor xenograft model. FIG. 33 a shows tumor volume of Balb/Cnu−/nu− mice injected with PPC-1 prostate cancer cells treated with TPI1396-22, TPI 1396-34 or solvent control. FIG. 33 b shows tumor weight ofmice sacrificed on day 19. Bars represent the median tumor size orweight.

FIG. 34 shows structures of agents TPI 1509-1 through TPI 1509-9 alongwith respective molecular weights, masses and lowest concentration ofeach agent having a ratio of 1.8.

FIGS. 35 A and B show compound modifications of R groups based on TPI1509-7 as an example of SAR studies for poly-phenylurea compounds.

FIG. 36A shows structures of TPI 1332-1 through TPI 1332-93 along withrespective molecular weights. FIG. 36B shows structures of selected TPI1332 compounds, including TPI 1332-4, TPI 1332-24, TPI 1332-41, TPI1332-69, TPI 1332-76, and TPI 1332-77 along with respective molecularweights and the activity of the compounds in competing with XIAP BIR2domain binding to the SMAC peptide. FIG. 36C shows the activity of1332-1 through TPI 1332-93 at 50 μg/ml in the derepression assay usingfull length XIAP. FIG. 36D shows the activity of TPI 1332-1 through TPI1332-93 at 16.7 μg/ml in the derepression assay using full length XIAP.FIG. 36E shows the activity of TPI 1332-1, -4, -41, -53, -69, and 77 inthe derepression assay using full length XIAP and the XIAP BIR2 domain.FIG. 36F shows the activity of TPI 1332-1, -2, -4, -6, -41, -47, -53,-55, -69, -76, -77 and -85 using cIAP BIR2 domain.

FIG. 37 shows that none of TPI 1495-1, -2, -3, -4, -6, -7, -8 or -9tetrapeptide series compete with XIAP binding to the SMAC peptide, whileTPI 1495-5 does compete with XIAP binding to the SMAC peptide; alsoshown is that TPI 1495-2, -3, -4, -6, -7, and -8 are inactive in thederepression assay using full length XIAP, while TPI 1495-1, -5, and -9are active in the derepression assay.

FIG. 38 shows that TPI 1396-34 functions by targeting XIAP protein.Cells from XIAP knock-out mice or wild type mice were treated witheither TPI 1396-34 or daunorubicin and % viability was assessed. Cellsused in these studies were either untransformed (FIGS. 38 A and C) ortransformed with a retrovirus encoding SC40 large T antigen (FIGS. 38 Band D).

FIG. 39 shows that TPI 1396-34 enhances cytotoxicity of antigen-specificCTL. Tumor cells treated with specific antigen were incubated withantigen-specific T cells at an effector:target ratio of either 5 (FIG.39B) or 10 (FIG. 39A). Percent cell lysis as a function of antigenconcentration is shown.

FIG. 40 shows that TPI 1396-12 effects in vivo activation of caspases.FIG. 40A shows an immunoblot of tumor tissue from animals treated withcontrol or TPI 1396-12, performed using antibodies specific for cleavedcaspase-3 or actin. FIG. 40B shows immunohistochemistry of tumor tissuesfrom animals treated with control or TPI 1396-12, performed usinghematoxylin and eosin (nuclear stain) (A and B); caspase-3 antibodiesand PCNA antibodies (C and D); caspase-6 antibodies (E and F) and DFF 40antibodies (G and H).

FIG. 41 shows toxicological analysis of TPI 1396-12, including whiteblood cell count (A); red blood cell count (B); platelet count (C); BUN(D); Bilirubin (E); ALT (F); and AST (G).

FIG. 42 shows that both TPI 1540-14 (a) and TPI 1540-15 (b) selectivelybinds to the BIR2 domain of XIAP, while inactive compound TPI 1540-20does not (c).

FIG. 43 shows the activity of TPI 1453-1 (also referred to as TPI 792-33or TPI 1408-3), TPI 1453-2, TPI 1453-3, TPI 1453-4, TPI 1453-5, TPI1453-6 (also referred to as TPI 792-35), TPI 1453-7, TPI 1453-8, and TPI1453-9, in the derepression assay using full length XIAP (XIAP-FLderepression data), as well as in the SMAC competition assay(Competitive binding assay).

FIG. 44 shows a table of biotinylated tetrapeptides of the TPI 1554series, as well as the corresponding peptide number for the originaltetrapeptides (Non-biotin Synthesis #) and molecular weights (MW).

FIGS. 45A-J show binding of BID and XIAP-BIR2 to biotinylated peptidesas follows: (A) TPI 1453-1 (TPI 1554-1); (B) TPI 1453-6 (TPI 1554-2);(C) TPI 1332-4 (TPI 1554-3); (D) TPI 1332-41 (TPI 1554-4); (E) TPI1332-69 (TPI 1554-5); (F) TPI 1332-77 (TPI 1554-6); (G) TPI 1495-19 (TPI1554-7); (H) TPI 1495-20 (TPI 1554-8); (1) SMAC 7-mer (TPI 1465-1, -2);and (J) SMAC 4-mer (TPI 1465-3, -4).

FIGS. 46 A-C show binding of three concentrations of XIAP-BIR2 tobiotinylated tetrapeptides, with FIG. 46A showing results using 1 μg/mlXIAP BIR2; FIG. 46B showing results using 0.5 μg/ml XIAP BIR2, and FIG.46C showing results using 0.25 μg/ml XIAP BIR2.

FIG. 47 shows competition of the binding of biotinylated tetrapeptideswith XIAP-BIR2, using (A) biotinylated TPI 1332-69, which is a non-SMACmimic; and (B) biotinylated TPI 1332-4, which is a SMAC mimic.

FIG. 48 shows binding of rhodamine labeled TPI-1332-4 (1566-11) toHis-BIR2 of XIAP and His-Traf2 (negative protein control). Rhodaminelabeled TPI 1332-4 was present at 2.4 μM in 50 mM KPi (potassiumphosphate) at pH 7.4/50 mM NaCl. His-BIR2 of XIAP and His-Traf2 werepresent at 0, 0.11, 0.33, 0.99, 2.96, 8.89, 26,67 and 80 μM. Plates wereincubated for 1 hour at room temperature and read in an LJL Analyst HTin fluorescence polarization mode with rhodamine filters (excitation 530nm; emission 580 nm) and a rhodamine dichroic mirror at 565 nm. Data wasfit in Prism™ by non-linear regression for a sigmoidal dose-responsecurve with variable slope.

FIG. 49 shows competitive binding of rhodamine labeled TPI 1332-4(1566-11) to HIS-BIR2 of XIAP in the presence of known IAP bindingcompound TPI 1396-11. Rhodamine labeled TPI 1332-4 was present at 2.4 μMand His-BIR2 of XIAP at 79 μM in 50 mM KPi at pH 7.4/50 mM NaCl. TPI1396-11 was at 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/ml. Plateswere incubated for 1 hour at room temperature and read in an LJL AnalystHT in fluorescence polarization mode with rhodamine filters (excitation530 nm; emission 580 nm) and a rhodamine dichroic mirror at 565 nm. Datawas fit in Prism™ by nonlinear regression for a sigmoidal dose-responsecurve with variable slope.

FIG. 50 shows binding of labeled TPI 1540-14 (TPI 1576-37, peaks 1 and2) to His-BIR1-2, His-Traf2 (negative protein control) and BIR1.Rhodamine labeled TPI 1540-14 (TPI 1576-37 pk1 or TPI 1576-37 pk2) werepresent at 2.5 μM in 50 mM Tris at pH 8.8/50 mM NaCl/1.25 mM DTT.His-BIR1-2 of XIAP, His-Traf2 and BIR1 of XIAP was present at 0, 0.14,0.41, 1.23, 3.70, 11.11, 33.33 and 100 μM. Plates were incubated for 1hour at room temperature and read in an LJL Analyst HT in fluorescencepolarization mode with rhodamine filters (excitation 530 nm; emission580 nm) and a rhodamine dichroic mirror at 565 nm. Data was fit inPrism™ by nonlinear regression for a sigmoidal dose-response curve withvariable slope.

FIG. 51 shows competitive binding of labeled TPI 1540-14 (TPI 1576-37,peak 2) to His-BIR1-2 against TPI 1396-11. TPI 1576-37 pk2 was presentat 2.5 μM and His-BIR1-2 of XIAP at 50 μM in 50 mM Tris at pH 8.8/50 mMNaCl/1.25 mM DTT. TPI 1396-11 was at 0, 1.56, 3.13, 6.25, 12.5, 25, 50and 100 μg/ml. Plates were incubated for 1 hour at room temperature andread in an LJL Analyst HT in fluorescence polarization mode withrhodamine filters (excitation 530 nm; emission 580 nm) and a rhodaminedichroic mirror at 565 nm. Data was fit in Prism by nonlinear regressionfor a sigmoidal dose-response curve with variable slope.

FIG. 52 shows binding of labeled TPI 1540 (TPI 1576-41, peaks 1 and 2)to His-BIR1-2, His-Traf2 (negative protein control) and BIR1. Rhodaminelabeled TPI 1540-14 (TPI 1576-41 pk1 or TPI 1576-41 pk2) were present at2.5 μM in 50 mM Tris at pH 8.8/50 mM NaCl/1.25 mM DTT. His-BIR1-2 ofXIAP, His-Traf2 and Bir1 of XIAP was present at 0, 0.14, 0.41, 1.23,3.70, 11.11, 33.33 and 100 M. Plates were incubated for 1 hour at roomtemperature and read in an LJL Analyst HT in fluorescence polarizationmode with rhodamine filters (excitation 530 nm; emission 580 nm) and arhodamine dichroic mirror at 565 nm. Data was fit in Prism by nonlinearregression for a sigmoidal dose-response curve with variable slope.

FIG. 53 shows competitive binding of labeled TPI 1540-14 (TPI 1576-41,peak 2) to His-BIR1-2 against TPI 1396-11. TPI 1576-41 pk2 was presentat 2.5 μM and His-BIR1-2 of XIAP at 50 μM in 50 mM Tris at pH 8.8/50 mMNaCl/1.25 mM DTT. TPI 1396-11 was at 0, 1.56, 3.13, 6.25, 12.5, 25, 50and 100 μg/ml. Plates were incubated for 1 hour at room temperature andread in an LJL Analyst HT in fluorescence polarization mode withrhodamine filters (excitation 530 nm; emission 580 nm) and a rhodaminedichroic mirror at 565 nm. Data was fit in Prism by nonlinear regressionfor a sigmoidal dose-response curve with variable slope.

FIG. 54 shows the aptoptotic effect of several compounds of theinvention. The apoptotic effect was determined as described in ExampleXXVIII.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides agents that suppress an inhibitor ofapoptosis protein (IAP) from inhibiting the protease activity of acaspase or from binding to a caspase. An advantage of an agent of theinvention is that it can be used to allow apoptosis to occur in a cellwhere apoptosis is being prevented by the regulatory activity of an IAP.Accordingly, the invention provides methods for reducing the ability ofa population of cells to survive in vitro or ex vivo by administering tothe cells an agent that derepresses an IAP-inhibited caspase. Use of anagent having specificity for a particular IAP-inhibited caspase in sucha method can selectively target and kill a sub-population of cells in alarger mixed population. Also provided is a method of treating anindividual having a condition characterized by a pathologically reducedlevel of apoptosis, such as cancer or hyperplasia, by administering tothe individual an agent of the invention, wherein the agent derepressesan IAP inhibited caspase, thereby increasing the level of apoptosis.

The invention further provides methods for identifying agents thatmodulate inhibitors of apoptosis. Using the methods of the invention acandidate agent can be tested for the ability to suppress an inhibitorof apoptosis (IAP) protein from inhibiting a protease activity of acaspase or from binding to a caspase. A caspase when uninhibitedmediates apoptosis. Thus, an agent determined by the methods toderepress an IAP-inhibited caspase is identified as an agent that allowsapoptosis to occur in the presence of negative regulatory components. Anadvantage of the methods of the invention is that they can be performedin a high throughput format such that large libraries of candidateagents can be efficiently screened for identification of a variety ofderepressors of an IAP-inhibited caspase.

As used herein the term “caspase” is intended to mean a member of thefamily of cysteine aspartyl-specific proteases that cleave C-terminal toan aspartic acid residue in a polypeptide and are involved in cell deathpathways leading to apoptosis. The term is intended to be consistentwith its use in the art as described, for example, in Martin and Green,Cell 82:349-352 (1995). The caspases previously were referred to as the“Ice” proteases, based on their homology to the first identified memberof the family, the interleukin-1β (IL-1β) converting enzyme (Ice), whichconverts the inactive 33 kiloDalton (kDa) form of L-1β to the active17.5 kDa form. The Ice protease was found to be homologous to theCaenorhabditis elegans ced-3 gene, which is involved in apoptosis duringC. elegans development, and transfection experiments showed thatexpression of Ice in fibroblasts induced apoptosis in the cells (seeMartin and Green, supra, 1995). Therefore, the term includes Ice andced-3.

Additional polypeptides sharing homology with Ice and ced-3 have beenidentified and are referred to as caspases, each caspase beingdistinguished by a number. For example, the originally identified Iceprotease now is referred to as caspase-1, the protease referred to ascaspase-3 previously was known variously as CPP32, YAMA and apopain, andthe protease now designated caspase-9 previously was known as Mch6 orICE-LAP6. The caspase family of proteases are characterized in that eachis a cysteine protease that cleaves C-terminal to an aspartic acidresidue and each has a conserved active site cysteine comprisinggenerally the amino acid sequence QACXG (SEQ ID NO:1), where X can beany amino acid and often is arginine. The caspases are furthersubcategorized into those that have DEVD (SEQ ID NO:2) cleavingactivity, including caspase-3 and caspase-7, and those that have YVAD(SEQ ID NO:3) cleaving activity, including caspase-1 (Martin and Green,supra, 1995).

As used herein the term “IAP” or “inhibitor of apoptosis protein” isintended to mean a protein that inhibits the proteolytic activity of acaspase. The term can include a protein that when bound to a caspaseinhibits the proteolytic activity of the caspase. The term can alsoinclude a protein that inhibits the proteolytic activity of a downstreamcaspase by inhibiting the ability of an upstream caspase to process aprecursor of the caspase to a mature form. Also included in the term isa protein that induces ubiquitination and degradation of a caspase.

Members of the Inhibitor of Apoptosis (IAP) protein family ofantiapoptotic proteins are conserved across evolution with homologuesfound in both vertebrate and invertebrate animal species. Thebaculovirus IAPs, Cp-IAP and Op-IAP, were the first members of thisfamily to be identified based on their ability to functionallycomplement defects in the cell death inhibitor p35, a baculovirusprotein that binds to and inhibits caspase. Subsequently, at least sevenadditional human homologues have been identified and demonstrated toinhibit cell death including X chromosome linked IAP (XIAP, GenBankaccession number U32974); cellular IAP proteins, c-IAP-1/HIAP-2/hMIHBand c-IAP-2/HIAP-1/hMIHC (Liston et al., Nature 379:349-353 (1996);Rothe et al., Cell 83:1243-1252 (1995)); neuronal apoptosis inhibitoryprotein, NAIP (Roy et al., Cell 80:167-178 (1995)); ML-IAP also referredto as LIVIN (Vucic et al., Cur. Biol. 10:1359-1366 (2000) and Kasof etal., J. Biol. Chem. 276:3238-3246 (2001)); Apollon (Chen et al.,Biochem. Biophys. Res. Commun. 264:847-854 (1999)); and survivin(Ambrosini et al., Nature Med. 3:917-921 (1997)). Two Drosophilahomologues (DIAP1 and DIAP2) have also been identified and demonstratedto inhibit cell death (Deveraux et al., Genes and Development 13:239-252(1999)). A central role for IAP-family proteins in programmed cell deathregulation in Drosophila has been suggested by the finding that severalapoptosis-inducing proteins in flies, including reaper, hid, and grimbind to IAPs as part of their cytotoxic mechanism. Other IAP proteinsinclude viral IAPs such as CiIAP, PoIAP, CpIAP and ASFIAP (Deveraux etal., supra (1999)).

AP proteins targeted by an agent of the invention include those thatinhibit the activity of an effector caspase such as caspase-3 orcaspase-7 and those that inhibit an initiator caspase such as caspase-9.The human IAPs (XIAP, cIAP1, and cIAP2) have been reported to bind andpotently inhibit caspase-3 and -7, with K_(i)s in the range of 0.2-10nM. These caspases operate in the distal portions of apoptotic proteasecascades, functioning as effectors rather than initiators of apoptosis.

A common structural feature of all IAP family members is a ˜70 aminoacid motif termed baculoviral IAP repeat (BIR), which is present in oneto three copies as described, for example, in Deveraux et al., Genes andDevelopment 13:239-252 (1999). The conserved presence and spacing ofcysteine and histidine residues observed within BIR domains indicatesthat the structure represents a zinc binding domain. BIR domains havebeen shown to exhibit distinct functions. For example, the second BIRdomain of XIAP (BIR2) is a potent inhibitor for caspase-3, whereas thethird BIR domain of XIAP (BIR3) targets caspase-9 (see Wu et al., Nature408:1008-1012 (2000)). In addition to the BIR motif located at theN-terminal and central portions of IAP, a RING finger domain is locatedin the C-terminal portion of members of the IAP protein family (Birnbaumet al., J. Virol. 68:2521-2528 (1994)). A BIR domain corresponds to anamino acid sequence having the consensus sequence:Xaa1-Xaa1-Xaa1-Arg-Xaa3-Xaa1-Xaa1-4-Xaa5-Xaa1-Xaa1-Trp-Xaa6-Xaa1-Xaa1-Xaa2-Xaa1-Xaa3-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Xaa3-Xaa3-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Xaa7-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa8-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Xaa5-Xaa3(SEQ ID NO: 16), wherein Xaa1 is any amino acid, Xaa2 is any amino acidor is absent, Xaa3 is a hydrophobic amino acid (for example, Ala, Cys,Ile, Leu, Met, Phe, Pro, Trp, Tyr, or Val), Xaa4 is serine or threonine,Xaa5 is phenylalanine or tyrosine, Xaa6 is proline or is absent, Xaa7 isaspartic or glutamic acid, and Xaa8 is a basic amino acid (for example,Arg, His, or Lys).

As used herein the term “IAP-inhibited caspase” is intended to mean acysteine aspartyl-specific protease that is prevented or suppressed fromproteolytic activity due to the presence of an inhibitor of apoptosisprotein. The term can include a cysteine aspartyl-specific proteasehaving reduced activity due to a bound inhibitor of apoptosis protein.The term can also include a cysteine aspartyl-specific protease that isprevented or suppressed from being processed to a mature form capable ofproteolytic activity due to the presence of an inhibitor of apoptosisprotein. An example of a non-processed cysteine aspartyl-specificprotease that is useful in the invention is a pro-caspase having anattached pro-domain. Alternatively, the compositions and methods of theinvention can be directed to an IAP-inhibited caspase that does notcontain a prodomain or is not a procaspase.

As used herein the term “derepress,” when used in reference to anIAP-inhibited caspase, is intended to mean reduction, inhibition orprevention of the ability of the IAP to inhibit the proteolytic activityof the caspase. Accordingly, a derepressor of a IAP-inhibited caspase isa molecule that inhibits or prevents the ability of the IAP to inhibitcaspase proteolytic activity. The term can include inhibition orprevention of the ability of an IAP to induce ubiquitination anddegradation of caspases.

As used herein, the term “agent” means a synthetic or isolatedbiological molecule such as a simple or complex organic molecule, apeptide, a peptidomimetic, a protein or an oligonucleotide that iscapable of derepressing an IAP-inhibited caspase.

As used herein, the term “pharmaceutically acceptable carrier” isintended to mean a medium having sufficient purity and quality for usein humans. Such a medium can be a human pharmaceutical grade, sterilemedium, such as water, sodium phosphate buffer, phosphate bufferedsaline, normal saline or Ringer's solution or other physiologicallybuffered saline, or other solvent or vehicle such as a glycol, glycerol,an oil such as olive oil or an injectable organic ester.Pharmaceutically acceptable media are substantially free fromcontaminating particles and organisms.

As used herein the term “inhibiting,” when used in reference to aprotein activity, is intended to mean a reduction in the activity bydecreasing affinity of the protein for a substrate or decreasing thecatalytic rate at which the protein converts a substrate to product. Theterm includes, for example, decreasing the affinity of an IAP for acaspase substrate, decreasing the affinity of a caspase for apolypeptide substrate, decreasing the rate at which a caspase cleaves apolypeptide C-terminal to an aspartic acid residue, or decreasing therate at which a caspase is ubiquitinated or proteolytically degraded.

As used herein the term “isolated,” when used in reference to an agent,means that the agent is separated from 1 or more reagent, precursor orother reaction product. Therefore, an isolated agent is an agent that isfree from one or more compounds found in the synthetic reaction orreaction pathway that produces the agent. Also included in the term isan agent that is free from one or more compound that it is found with innature. An isolated agent also includes a substantially pure agent. Theterm can include a molecule that has been produced by a combinatorialchemistry method and separated from precursors and other products bychemical purification or by binding to second molecule with sufficientstability to be co-purified with the second molecule. The term caninclude naturally occurring agents such as products of biosyntheticreactions or non-naturally occurring agents.

As used herein the term “peptide” refers to a molecule containing two ormore amino acids linked by a covalent bond between the carboxyl of oneamino acid and the amino group of another. Invention peptides can beincluded in larger molecules or agents, such as larger peptides,proteins, fragments of proteins, peptoids, peptidomimetics and the like.A peptide can be a non-naturally occurring molecule, which does notoccur in nature, but is produced as a result of in vitro methods, or canbe a naturally occurring molecule such as a protein or fragment thereofexpressed from a cDNA library. Peptides can be either linear, cyclic ormultivalent, and the like, which conformations can be achieved usingmethods well-known in the art. The term includes molecules havingnaturally occurring proteogenic amino acids as well as non-naturallyoccurring amino acids such as D-amino acids and amino acid analogs, anyof which can be incorporated into a peptide using methods known in theart. In view of this definition, one skilled in the art would know thatreference herein to an amino acid, unless specifically indicatedotherwise, includes, for example, naturally occurring proteogenicL-amino acids, D-amino acids, chemically modified amino acids such asamino acid analogs, naturally occurring non-proteogenic amino acids suchas norleucine, and chemically synthesized agents. Exemplary amino acidsuseful in the invention are described further below.

As used herein, the term “proteogenic,” when used in reference to anamino acid, indicates that the amino acid can be incorporated into aprotein in a cell through well known metabolic pathways. The amino acidsare designated as D or L in reference to the configuration at the alphacarbon. Amino acids referred to herein without specific reference toconfiguration are understood to have the L configuration at the alphacarbon. Proteogenic amino acids are indicated herein using the singleletter or three letter code and are intended to be consistent with thenomenclature used in the art as described for Example in Branden andTooze Introduction to Protein Structure, Garland Publishing, New York,pp 6-7 (1991). Other amino acids are indicated using nomenclature knownin the art, wherein, for example, pClPhe refers top-chloro-phenylalanine, ThiAla refers to 2-thienyl-alanine, Nal refersto 3-(2-napthyl)-alanine, 3I-Tyr refers to 3-iodo-Tyrosine, Cha refersto cyclohexylalanine, Lys-e-Fmoc refers tolysine(e-fluorenylmethloxycarbonyl) and OEt-Tyr refers toTyrosine(O-ethyl).

As used herein the term “core” is intended to mean a chemical structureor motif of a molecule, or portion thereof. The chemical structure ormotif can be, for example, an amino acid sequence of a peptide orpeptide containing molecule, or a chemical formula representing thecovalent attachment of atoms in a molecule. A chemical structure ormotif included in the term can be further defined with respect tochirality. A core peptide or other chemical entity need not be locatedat the center of a molecule.

The present invention provides isolated agents that derepresses anIAP-inhibited caspase. An agent that derepresses an IAP-inhibitedcaspase can have a core peptide or amino acid sequence motifcorresponding to:

-   -   (L-Ala)-X₁-(L-Trp)-X₂ (Core peptide 4)        where X₁ is L-Trp or D-Trp and X₂ is L-ThiAla or L-pClPhe.        Exemplary core peptides included in Core peptide 4 include, for        example:    -   (L-Ala)-(L-Trp)-(L-Trp)-(L-ThiAla) (Core peptide 5),    -   (L-Ala)-(L-Trp)-(L-Trp)-(L-pClPhe)(Core peptide 6) and    -   (L-Ala)-(D-Trp)-(L-Trp)-(L-ThiAla) (Core peptide 7).

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   X₁-X₂-X₃-X₄ (Core peptide 23)        where X₁ is L-Ala, L-Cha, L-Nal, D-Trp or D-Trp(CHO); X₂ is        D-Nal, D-Tip, D-Trp(CHO), L-Trp, L-Trp(CHO), D-Cha or D-ThiAla;        X₃ is L-Trp, L-Trp(CHO) or D-Phe; and X₄ is L-Nal, D-Nal, D-Trp,        D-Trp(CHO), L-ThiAla, L-3I-Tyr or L-pClPhe. Exemplary core        peptides included in Core peptide 23 include, for example:    -   (L-Ala)-(D-Nal)-(L-Trp)-(L-Nal) (Core peptide 24)    -   (D-Trp)-(D-Trp)-(L-Trp)-(D-Nal) (Core peptide 25)    -   (L-Cha)-(D-Nal)-(L-Trp)-(L-ThiAla) (Core peptide 26)    -   (L-Ala)-(L-Trp)-(L-Trp)-(L-3I-Tyr) (Core peptide 27)    -   (L-Ala)-(D-Trp)-(L-Trp)-(L-ThiAla) (Core peptide 28)    -   (L-Cha)-(L-Trp)-(L-Trp)-(L-pClPhe) (Core peptide 29)    -   (L-Ala)-(D-Trp)-(L-Trp)-(D-Trp) (Core peptide 30)    -   (L-Ala)-(D-Trp)-(D-Phe)-(D-Trp) (Core peptide 31)    -   (L-Nal)-(D-Trp)-(D-Phe)-(D-Trp) (Core peptide 32)    -   (L-Nal)-(D-Cha)-(L-Trp)-(D-Trp) (Core peptide 33)    -   (L-Nal)-(D-ThiAla)-(D-Phe)-(D-Trp) (Core peptide 34).

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   X₁-X₂-X₃-X₄ (Core peptide 8)        where X₁ is D-Nal or L-ThiAla; X₂ is Lys-εFmoc, D-pClPhe or        L-Nal; X₃ is D-Nal, L-pClPhe or D-Lys(Fm); and X₄ is Lys-εFmoc        or D-pFPhe. Exemplary core peptides included in Core peptide 8        include, for example:    -   (D-Nal)-(Lys-εFmoc)-(L-pClPhe)-(Lys-εFmoc) (Core peptide 9)    -   (D-Nal)-(D-pClPhe)-(L-pClPhe)-(Lys-εFmoc) (Core peptide 10)    -   (D-Nal)-(L-Nal)-(L-pClPhe)-(Lys-εFmoc) (Core peptide 11)    -   (D-Nal)-(L-Nal)-(D-Lys-εFmoc)-(Lys-εFmoc) (Core peptide 12)    -   (L-ThiAla)-(Lys-εFmoc)-(D-Nal)-(Lys-εFmoc) (Core peptide 13)    -   (L-ThiAla)-(Lys-εFmoc)-(L-pClPhe)-(pF-D-F) (Core peptide 14)    -   (L-ThiAla)-(D-pClPhe)-(L-pClPhe)-(Lys-εFmoc) (Core peptide 15)    -   (L-ThiAla)-(L-Nal)-(L-pClPhe)-(Lys-εFmoc) (Core peptide 16)    -   (L-ThiAla)-(L-Nal)-(D-Lys-εFmoc)-(Lys-εFmoc) (Core peptide 17).

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   X₁-X₂-X₃-X₄ (Core peptide 35)        where X₁ is L-ThiAla or Phe; X₂ is D-pClPhe or D-OEt-Tyr; X₃ is        D-Nal, or D-OEt-Tyr; and X₄ is D-pClPhe or D-pNO₂Phe. Exemplary        core peptides included in Core peptide 35 include, for example:    -   (L-ThiAla)-(D-pClPhe)-(D-Nal)-(D-pClPhe) (Core peptide 36)    -   (L-ThiAla)-(D-pClPhe)-(D-Nal)-(D-pNO₂Phe) (Core peptide 37)    -   (L-ThiAla)-(D-OEt-Tyr)-(D-OEt-Tyr)-(D-pClPhe) (Core peptide 38)    -   (Phe)-(D-pClPhe)-(D-Nal)-(D-pClPhe) (Core peptide 39).

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   A-X₁-X₂-X₃ (Core peptide 18)        where X₁ is Met, Ser, Thr, Trp, or ThiAla and X₂ and X₃ are        selected from Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,        Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, D-Ala, D-Asp,        D-Glu, D-Phe, D-His, D-Ile, D-Lys, D-Leu, D-Met, D-Asn, D-Pro,        D-Gln, D-Arg, D-Ser, D-Thr, D-Val, D-Trp, D-Tyr, L-Nle, D-Nle,        L-Cha, D-Cha, L-PyrAla, D-PyrAla, L-ThiAla, D-ThiAla, L-Tic,        D-Tic, L-pClPhe, D-pClPhe, L-pIPhe, D-pIPhe, L-pNO₂Phe,        D-pNO₂Phe, L-Nal, D-Nal, beta-Ala, e-Aminocaproic acid,        L-Met[O₂], L-dehydPro, or L-3I-Tyr.

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   X₁-X₂-(L-Trp)-(D-Trp) (Core peptide 19)        where X₁ and X₂ are selected from Ala, Asp, Glu, Phe, Gly, His,        Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr,        D-Ala, D-Asp, D-Glu, D-Phe, D-His, D-Ile, D-Lys, D-Leu, D-Met,        D-Asn, D-Pro, D-Gln, D-Arg, D-Ser, D-Thr, D-Val, D-Trp, D-Tyr,        L-Nle, D-Nle, L-Cha, D-Cha, L-PyrAla, D-PyrAla, L-ThiAla,        D-ThiAla, L-Tic, D-Tic, L-pClphe, D-pClPhe, L-pIPhe, D-pIPhe,        L-pNO₂Phe, D-pNO₂Phe, L-Nal, D-Nal, beta-Ala, e-Aminocaproic        acid, L-Met[O₂], L-dehydPro, or L-3I-Tyr.

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to:

-   -   X₁-X₂-X₃-X₄-W-W (Core peptide 55),        where X₁, X₂ and X₃ are selected from Ala, Asp, Glu, Phe, Gly,        His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,        Cys or Tyr and X₄ is selected from Ala, His, Lys, Asn, Gln, Arg,        Ser, Thr or Val.

An agent that derepresses an IAP-inhibited caspase can have a corepeptide or amino acid sequence motif corresponding to any of:

-   -   X₁-X₂-A-A-W-W (Core peptide 43), SEQ ID NO: 7    -   X₁-X₂-G-A-W-W (Core peptide 44), SEQ ID NO: 8    -   X₁-X₂-R-A-W-W (Core peptide 45), SEQ ID NO: 9    -   X₁-X₂-X₄-A-W-W (Core peptide 46),    -   X₁-X₂-C-K-W-W (Core peptide 47), SEQ ID NO: 10    -   X₁-X₂-L-X₃-W-W (Core peptide 20),    -   X₁-X₂-R-X₃-W-W (Core peptide 21),    -   X₁-X₂-G-X₃-W-W (Core peptide 22),    -   X₁-X₂-T-X₃-W-W (Core peptide 42),    -   X₁-X₂-V-X₃-W-W (Core peptide 48),    -   X₁-T-X₂-X₃-W-W (Core peptide 49),    -   X₁-Y-X₂-X₃-W-W (Core peptide 50),    -   A-X₁-X₂-X₃-W-W (Core peptide 51),    -   C-X₁-X₂-X₃-W-W (Core peptide 52),    -   F-X₁-X₂-X₃-W-W (Core peptide 53), or    -   K-X₁-X₂-X₃-W-W (Core peptide 54),        where X₁, X₂ and X₄ are selected from Ala, Asp, Glu, Phe, Gly,        His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,        Cys or Tyr and X₃ is selected from Ala, Lys, Ser or Thr.

The core peptide sequences of the invention can be those of a moleculeor a portion of a molecule. For example, the above-described sequenceshaving four positions can be tetrapeptide molecules and theabove-described sequences having four or six positions can behexapeptide molecules. A core peptide of the invention can also beincluded in larger molecules including, for example, a molecule havingat least 5 amino acids, at least 6 amino acids, at least 7 amino acids,at least 8 amino acids, at least 9 amino acids, at least 10 amino acids,at least 20 amino acids or at least 25 amino acids. In some embodiments,the amino acid lengths of molecules comprising invention peptides can bedefined by a maximum length including, for example, no more than about4, no more than about 5, no more than about 6, no more than about 7, nomore than about 8, no more than about 9, no more than about 10, no morethan about 20, no more than about 25, no more than about 50, no morethan about 100, no more than about 150, or no more than about 200 ormore amino acids in length so long as the peptide is capable ofderepressing an IAP-inhibited caspase. A molecule having a core peptideof the invention can also be defined within a size range delimited by acombination of any of the above described minimum and maximum lengths.

The invention further provides agents that are effective derepressors ofan IAP-inhibited caspase having non-peptide based core structures. Thus,the invention provides an agent that derepresses an IAP-inhibitedcaspase and having a core structure corresponding to anN-benzyl-1,4,5-trisubstituted-2,3-diketopiperazine such as TPI 759 shownin FIG. 8. An agent having the TPI 759 core structure can besubstituted, for example, at position R1 derived from an amino acid sidechain group of norleucine, NapAla, cyclohexylalanine, Lys, norvaleucineor valine; at R2 derived from an amino acid side chain group of Leu,NapAla, Phe, Ile or Val; and at R3 with the functional group derivedfrom 4-isobutyl-alpha-methylphenylacetic acid,3,5-bis(trifluoromethyl)-phenylacetic acid, heptanoic acid,(alpha-alpha-alpha-trifluoro-m-tolyl)acetic acid,4-tert-butyl-cyclohexane carboxylic acid, m-tolylacetic acid,3,4-dichlorophenylacetic acid, 3,3-diphenyl propionic acid,dicyclohexylacetic acid, cycloheptanecarboxylic acid, p-Tolylacetic acidor cyclohexanebutyric acid as shown in FIG. 8.

An agent that derepresses an IAP-inhibited caspase can have a corestructure corresponding to a C-6-acylamino bicyclic guanidine such asTPI 882 shown in FIG. 7. An agent having the TPI 882 core structure canbe substituted, for example, at position R1 derived from an amino acidside chain group of L-cyclohexylalanine, D-cyclohexylalanine,D-2-chloroPhe, O-ethyl-D-Tyr, p-iodo-L-Phe, p-iodo-D-Phe, D-homo-Phe,L-homo-Phe, L-napthylAla, D-napthylAla or L-4,4-biphenylalanine; atposition R2 with the functional group derived from 2-phenylbutyric acid,3-phenylbutyric acid, m-tolylacetic acid, 3-fluorophenylacetic acid,p-tolylacetic acid, 4-fluorophenylacetic acid, 3-methoxyphenylaceticacid, 4-methoxyphenylacetic acid, 4-ethoxyphenylacetic acid,4-biphenylacetic acid, phenylacetic acid, 4-phenylbutyric acid,heptanoic acid, 4-methylvaleric acid, tert-butyric acid,cyclohexylcarboxylic acid, cyclohexylacetic acid, cyclohexylbutyricacid, cycloheptanecarboxylic acid, cyclobutanecarboxylic acid,cyclopentylcarboxylic acid, 3-cyclopentylpropionic acid,cyclohexylpropionic acid, 4-methyl-1-cyclohexylcarboxylic acid,4-t-butylcyclohexylcarboxylic acid, 2-norbornaneacetic acid,1-adamantane acetic acid, 2-ethylbutyric acid, 3,3-diphenylpropionicacid or cyclopentylacetic acid; and at position R3 with the functionalgroup derived from 3-fluorophenylacetic acid, 4-ethoxyphenylacetic acid,4-biphenylacetic acid or 3,5-bis(trifluoromethyl)phenylacetic acid asshown in FIG. 7. An agent having the TPI 882 core structure can besubstituted at R2 and R3 with the functional group derived fromphenylacetic acid and at R1 derived from an amino acid side chain groupof L-cyclohexylalanine, D-cyclohexylalanine, D-p-chloro-Phe,D-p-fluoro-Phe, L-p-fluoro-Phe, D-2-chloro-Phe, O-ethyl-L-Tyr,O-ethyl-D-Tyr, O-methyl-D-Tyr, 3,5-diiodo-Tyr or L-napthylAla; at R1with an amino acid side chain group of Phe; at R3 with the functionalgroup derived from phenylacetic acid and at R2 with the functional groupderived from p-tolylacetic acid, 4-fluorophenylacetic acid,3-methoxyphenylacetic acid, 4-methoxyphenylacetic acid,4-ethoxyphenylacetic acid, 4-biphenylacetic acid, phenylacetic acid,4-phenylbutyric acid, heptanoic acid, 3-methylvaleric acid or4-methylvaleric acid; or at R1 with an amino acid side chain group ofPhe; at R2 with the functional group derived from phenylacetic acid; andat R3 with the functional group derived from 4-biphenylacetic acid,cyclohexanecarboxylic acid, cyclohexylacetic acid, cyclohexylbutyricacid, cycloheptanecarboxylic acid, 3-cyclopentylpropionic acid or3,5-bis(trifluoromethyl)phenylacetic acid as shown in FIG. 10.

An agent that derepresses an IAP-inhibited caspase can have a corestructure corresponding to a polyphenylurea such as TPI 927 shown inFIG. 6. An agent having the TPI 927 core structure can be substituted,for example, at position R1 derived from an amino acid side chain groupof D-Lys(Me), L-3-(2Nap)Ala, D-Chala, L-Phe, Pro, Leu or Ser; atposition R2 derived from an amino acid side chain group of ε-Lys, L-Nle,D-Phe, Pro, D-Orn(Me), Gln, L-3-(2-Nap)Ala or D-Thr; and at position R3with the functional group derived from 4-methoxyphenylacetic acid,1-adamantaneacetic acid, cyclohexanebutyric acid,4-tert-butylcyclohexanecarboxylic acid, cycloheptanecarboxylic acid,3-fluorophenylacetic acid, 3,3-diphenylpropionic acid,4-ethoxyphenylacetic acid, 1-phenyl-1-cyclopropanecarboxylic acid,1-napthylacetic acid, or cyclobutane carboxylic acid as shown in FIG. 6.An agent having the TPI 927 core structure can be substituted at R1 andR2 with an amino acid side chain group of Phe and at R3 with thefunctional group derived from trimethylacetic acid, hydrocinnamic acid,4-tert-butylcyclohexane carboxylic acid,4-methyl-1-cyclohexanecarboxylic acid, cyclopentylacetic acid,1-phenyl-1-cyclopropanecarboxylic acid, cyclohexanecarboxylic acid,phenylacetic acid, cycloheptanecarboxylic acid, cyclobutane carboxylicacid, cyclohexanebutyric acid, 1-adamantaneacetic acid,cyclopentanecarboxylic acid, isobutyric acid, cyclohexylacetic acid;3-methoxyphenylacetic acid, butyric acid,3-(3,4,5)-trimethoxyphenylpropionic acid; heptanoic acid;2-norbomaneacetic acid, cyclohexanepropionic acid, tert-butyric acid,4-ethoxyphenylacetic acid, 3,3-diphenylpropionic acid,4-methoxyphenylacetic acid, acetic acid, methylvaleric acidp-tolylacetic acid or 4-isobutyl-alpha-methylphenylacetic acid as shownin FIG. 9.

An agent that derepresses an IAP-inhibited caspase can have a corestructure corresponding to an N-acyltriamine such as TPI 914 shown inFIG. 4. An agent having the TPI 914 core structure can be substituted,for example, at position R1 derived from an amino acid side chain groupof Nap-Ala or 4-Fluoro-phenylalanine; at position R2 derived from anamino acid side chain group of L-Trp, Nap-Ala, D-Trp,4-chlorophenylalanine, D-cyclohexylalanine or Tyr; and at R3 with thefunctional group derived from 4-vinylbenzoic acid,4-ethyl-4-biphenylcarboxylic acid, 3,5-Bis(trifluoromethyl)-phenylaceticacid, 4-biphenylcarboxylic acid, 4-biphenylacetic acid or3,5-bis-(trifluoromethyl)-benzoic acid as shown in FIG. 4. An agenthaving the TPI 914 core structure can be substituted at R1 with afunctional group derived from an amino acid side chain group of Leu, atR2 with a functional group derived from an amino acid side chain groupof D-Trp and at R3 with methyl; at R1 with a functional group derivedfrom an amino acid side chain group of Leu, at R2 with a functionalgroup derived from an amino acid side chain group of Phe and at R3 withthe functional group derived from 3,5-Bis(trifluoromethyl)-phenylaceticacid; at R1 with a functional group derived from an amino acid sidechain group of Leu, at R2 with a functional group derived from an aminoacid side chain group of Phe and at R3 with the functional group derivedfrom 4-vinylbenzoic acid; or at R1 with a functional group derived froman amino acid side chain group of Leu, at R2 with a functional groupderived from an amino acid side chain group of Phe and at R3 with thefunctional group derived from 4-ethyl-4-biphenylcarboxylic acid each asshown in FIG. 5.

Those skilled in the art will recognize that libraries having the corestructure of TPI 914, TPI 927, TPI 759, TPI 882, can becombinatorialized at one or more position. A combinatorialized positionrefers to a position which is variously substituted with differentmoieties such that a library of molecules combinatorialized at theposition is a mixture of molecules that differ in chemical structure atthat position. Such libraries can be used to identify agents thatderepress an IAP-inhibited caspase, for example, in a screen utilizingpositional scanning as described in Example VI. Thus, any one ofpositions R1, R2 or R3 can be held fixed to a discrete moiety while theremaining two positions are combinatorialized, thereby generatingsublibraries based on which position is fixed. Moreover, one can addadditional positions to the core structure that can be combinatorializedor held constant while one or more other positions arecombinatorialized. Thus, different or more diverse libraries can becreated based on a particular core structure or on a species identifiedfrom the library as capable of derepressing an IAP-inhibited caspase.

Those skilled in the art will understand that an agent of the inventionhaving a core structure corresponding to TPI 914, TPI 927, TPI 759, TPI882, such as a compound of the TPI 1396, TPI 1349, TPI 1391 or TPI 1400series, can further include one or more attached moieties such as apeptide moiety. An agent of the invention can be multivalent, asdescribed above, in which case the attached moiety can be one or morecore structures corresponding to TPI 914, TPI 927, TPI 759, TPI 882, acore peptide having a sequence described above; or a combination of oneor more of these core structures and core peptides.

An agent that is capable of derepressing an IAP-inhibited caspase,whether based on a peptide or non-peptide core structure can include amoiety known to naturally occur in biological proteins. Such moietieswhen part of a protein are commonly referred to as amino acid R-groups.These R groups can be characterized by a variety of physical or chemicalproperties. Taking the essential amino acids as an example, the R groupsfound on Gly, Ala, Val, Leu, or Ile have the characteristic of beingnon-polar; polar R groups include the sulfhydryl moiety of Cys, thethioether of Met, hydroxyl moieties of Ser and Thr, and amide moietiesof Asn and Gln; Asp and Glu are characterized as polar acidic groups dueto the presence of carboxylic acid moieties; polar basic R groupsinclude Lys which has an amino moiety, Arg which has a guanidino moietyand His which has an imidazole with secondary amines; and Phe, Trp, Tyr,and His are characterized as aromatic amino acids due to the presence ofphenyl or heterocyclic rings. An agent of the invention can include oneor more of these moieties or characteristics, thereby rendering theagent capable of derepressing an IAP-inhibited caspase.

An agent of the invention can also be described or characterizedaccording to other moieties or combinations of moieties that whenpresent renders the agent capable of derepressing an IAP-inhibitedcaspase. Definitions for various moieties that can be present in theagents of the invention are set forth below.

As used herein, the term “alkyl,” alone or in combination, refers to asaturated, straight-chain or branched-chain hydrocarbon moietycontaining from 1 to 10, preferably from 1 to 6 and more preferably from1 to 4, carbon atoms. Examples of such moieties include, but are notlimited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the like.

The term “alkene,” alone or in combination, refers to a straight-chainor branched-chain hydrocarbon moiety having at least one carbon-carbondouble bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such moieties include,but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- andZ-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl, methylidene(═CH₂), ethylidene (—CH═CH—), propylidene (—CH₂—CH═CH—) and the like.

The term “alkyne,” alone or in combination, refers to a straight-chainor branched-chain hydrocarbon moiety having at least one carbon-carbontriple bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such moieties include,but are not limited to, ethynyl (acetylenyl), propynyl (propargyl),butynyl, hexynyl, decynyl and the like.

The term “cycloalkyl,” alone or in combination, refers to a saturated,cyclic arrangement of carbon atoms which number from 3 to 8 andpreferably from 3 to 6, carbon atoms. Examples of such cycloalkylmoieties include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term “aryl” refers to a carbocyclic (consisting entirely of carbonand hydrogen) aromatic group selected from the group consisting ofphenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, andanthracenyl; or a heterocyclic aromatic group selected from the groupconsisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl,imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl,pyridazinyl, pyrimidinyl. pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl,indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzo[b]thiophenyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl.

“Aryl” groups, as defined in this application may independently containone to four substituents which are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, amino, nitro,trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano,carboxy, carboalkoxy, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy,alkylamino, alkenylamino, alkynylamino, aliphatic or aromatic acyl,alkoxy-carbonylamino, alkylsulfonylamino, morpholinocarbonylamino,thiomorpholinocarbonylamino, N-alkyl guanidino, aralkylaminosulfonyl;aralkoxyalkyl; N-aralkoxyurea; N-hydroxylurea; N-alkenylurea;N,N-(alkyl, hydroxyl)urea; heterocyclyl; thioaryloxy-substituted aryl;N,N-(aryl, alkyl)hydrazino; Ar′-substituted sulfonylheterocyclyl;aralkyl-substituted heterocyclyl; cycloalkyl and cycloakenyl-substitutedheterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl;heterocyclylamino; aliphatic or aromatic acylaminocarbonyl; aliphatic oraromatic acyl-substituted alkenyl; Ar′-substituted aminocarbonyloxy;Ar′, Ar′-disubstituted aryl; aliphatic or aromatic acyl-substitutedacyl; cycloalkylcarbonylalkyl; cycloalkyl-substituted amino;aryloxycarbonylalkyl; phosphorodiamidyl acid or ester;

“Ar′” is a carbocyclic or heterocyclic aryl group as defined abovehaving one to three substituents selected from the group consisting ofhydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl,trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene,1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylaminoor alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl,alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl orN,N-dialkyl urea.

The term “alkoxy,” alone or in combination, refers to an alkyl ethermoiety, wherein the term “alkyl” is as defined above. Examples ofsuitable alkyl ether moieties include, but are not limited to, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy and the like.

The term “alkenoxy,” alone or in combination, refers to a moiety offormula alkenyl-O—, wherein the term “alkenyl” is as defined above.Examples of suitable alkenoxy moieties include, but are not limited to,allyloxy, E- and Z-3-methyl-2-propenoxy and the like.

The term “thioalkoxy” refers to a thioether moiety of formula alkyl-S—,wherein alkyl is as defined above.

The term “alkylamino,” alone or in combination, refers to a mono- ordi-alkyl-substituted amino group (i.e., a group of formula alkyl-NH- or(alkyl)₂-N—), wherein the term “alkyl” is as defined above. Examples ofsuitable alkylamino moieties include, but are not limited to,methylamino, ethylamino, propylamino, isopropylamino, t-butylamino,N,N-diethylamino and the like.

The term “amide” refers to either —N(R¹)—C(═O)— or —C(═O)—N(R¹)— where(R¹) is defined herein to include hydrogen as well as other groups. Theterm “substituted amide” refers to the situation where (R¹) is nothydrogen, while the term “unsubstituted amide” refers to the situationwhere (R¹) is hydrogen.

The term “aryloxy,” alone or in combination, refers to a moiety offormula aryl-O—, wherein aryl is as defined above. Examples of aryloxymoieties include,

but are not limited to, phenoxy, naphthoxy, pyridyloxy and the like.

The term “arylamino,” alone or in combination, refers to a moiety offormula aryl-NH—, wherein aryl is as defined above. Examples ofarylamino moieties include, but are not limited to, phenylamino(anilido), naphthylamino, 2-, 3- and 4-pyridylamino and the like.

The term “aryl-fused cycloalkyl,” alone or in combination, refers to acycloalkyl moiety which shares two adjacent atoms with an aryl moiety,wherein the terms “cycloalkyl” and “aryl” are as defined above. Anexample of an aryl-fused cycloalkyl moiety is a benzofused cyclobutylgroup.

The term “alkylcarbonylamino,” alone or in combination, refers to amoiety of formula alkyl-CONH, wherein the term “alkyl” is as definedabove.

The term “alkoxycarbonylamino,” alone or in combination, refers to amoiety of formula alkyl-OCONH—, wherein the term “alkyl” is as definedabove.

The term “alkylsulfonylamino,” alone or in combination, refers to amoiety of formula alkyl-SO₂NH—, wherein the term “alkyl” is as definedabove.

The term “arylsulfonylamino,” alone or in combination, refers to amoiety of formula aryl-SO₂NH—, wherein the term “aryl” is as definedabove.

The term “N-alkylurea,” alone or in combination, refers to a moiety offormula alkyl-NH—CO—NH—, wherein the term “alkyl” is as defined above.

The term “N-arylurea,” alone or in combination, refers to a moiety offormula aryl-NH—CO—NH—, wherein the term “aryl” is as defined above.

The term “halogen” means fluorine, chlorine, bromine and iodine.

In view of the above definitions, other chemical terms used throughoutthis application can be easily understood by those of skill in the art.Terms may be used alone or combined to describe a combination ofmoieties according to accepted chemical nomenclature.

An agent of the invention can be synthesized using reagents andconditions well known to yield products having predictable moieties orcharacteristics. For example, peptides can be synthesized in largenumbers at relatively low cost and they can be readily modified toexhibit diverse properties (see, for example, Rees et al., ProteinEngineering: A Practical Approach (IRL Press 1992)). A peptidederepressor of an IAP-inhibited caspase can be synthesized using amodification of the solid phase peptide synthesis method (Merrifield (J.Am. Chem. Soc., 85:2149 (1964); Houghten, U.S. Pat. No. 4,631,211,issued Dec. 23, 1986) or can be synthesized using standard solutionmethods well known in the art (see, for example, Bodanszky, M.,Principles of Peptide Synthesis 2nd ed. (Springer-Verlag, 1988 and 1993,suppl.)). Peptides prepared by the method of Merrifield can besynthesized using an automated peptide synthesizer such as the AppliedBiosystems 431A-01 Peptide Synthesizer (Mountain View, Calif.) or usinga manual peptide synthesis method (Houghten, supra, 1986).

Furthermore, combinatorial methods such as those described below can beused to make an agent that derepresses an IAP-inhibited caspase. Alibrary can be synthesized to have candidate agents with particularmoieties such as those defined above or described in the Examples setforth below. Additionally, the synthetic conditions can be selected toproduce a library of candidate compounds with particular characteristicsinherent in one or more of the moieties described herein such as thecharacteristics described above for amino acid R groups. For example, alibrary can be synthesized to have characteristics of SMAC a naturallyoccurring IAP inhibitor. The N-terminal region of SMAC has been shown tomediate binding to and inhibition of IAPs (see Srinivasula et al.,Nature 410:112-116 (2001), Wu et al., Nature 408:1008-1012 (2000), Liuet al., Nature 408:1004-1008 (2000)). Accordingly, this N-terminaldomain can be used to guide library synthesis such that reactants andconditions are chosen to selectively incorporate similar moieties andcharacteristics into the candidate agents in the library. Similarstrategies can be employed using agents described herein or identifiedby the methods of the invention, wherein a library is made toselectively contain characteristics or moieties found in a particularagent or common to a plurality of agents. Such a design strategyincreases the probability that an effective derepressor of anIAP-inhibited caspase will be identified.

An agent of the invention that is capable of derepressing anIAP-inhibited caspase can be identified in a screen or otherwisecharacterized according to any of a variety of functional propertiesdescribed herein. In one embodiment a derepressor of an IAP-inhibitedcaspase is identified or otherwise characterized based on its ability toallow caspase activity in the presence of an IAP. For example, theeffectiveness of a compound of the invention can be determined accordingto the ratio of caspase activity for an IAP-inhibited caspase in thepresence and absence of an agent of the invention.

Using caspase derepression assays, several compounds have been disclosedherein that derepress an IAP-inhibited caspase. For example, theinvention provides an isolated agent having a core structure selectedfrom any of the structures shown in FIGS. 21-24, where the agent isselected from TPI 1349-1 through 1349-34; TPI 1396-1 through TPI1396-36; TPI 1391-1 through TPI 1391-36; and TPI 1400-1 through TPI1400-58 and where the agent derepresses an IAP-inhibited caspase.

A compound of the invention that derepress an IAP-inhibited caspase canbe a member of a disclosed compound class, such as a polyphenylurea,diketopiperazine, bicyclic guanidine, N-acyl triamine, or atetrapeptide. A summary of various activities observed for compoundclasses disclosed herein is presented in Table XII, below. This tableshows average activities of representative compounds from thepolyphenylurea, diketopiperazine, bicyclic guanidine, N-acyl triamine,and tetrapeptide classes in the caspase derepression assay, SMACcompetition assay and the Jurkat cell cytotoxicity assay. Polyphenylureaand diketopiperazines were found to have activity in the enzymederepression assay in the presence of either full length XIAP or XIAPBIR2 domain, as is described in Example VIII. TABLE XII IAP AntagonistsFamilies of Compounds Enzyme Derepress SMAC Cell Activity Compound ClassIC-50 (μM) competition IC-50 (μM) Poly-phenylurea 12 No 7Diketopiperazines 32 No 8 Bicyclic guanidines 32 N.T. N.T. N-Acyltriamines 53 N.T. N.T. Tetrapeptides-1 19 No 8 Tetrapeptides-2 9 Yes 40

An exemplary assay for identifying a compound that derepresses anIAP-inhibited caspase is provided in Example I and use of the assay toidentify such derepressor compounds is demonstrated in Examples Ithrough VI. As described below in the Examples, a ratio of V_(max) inthe presence and absence of the agent for an IAP-inhibited caspase thatis at least about 1.7, depending upon assay conditions, is indicative ofan effective derepressor of an IAP-inhibited caspase. Those skilled inthe art will understand that a value for this ratio that is indicativeof effectiveness will depend upon the concentration of the agent usedand the IC₅₀ of the agent. Accordingly, when higher concentrations ofthe agent are used the threshold value for the ratio of V_(max) in thepresence and absence of the agent can be at least about 2 at least about2.5, at least about 3 or at least about 4 or higher. When lowerconcentrations of the agent are used this ratio can be as low as atleast about 1.5, at least about 1.3 at least about 1 or lower. Thus, itcan be appropriate to express the ratio in combination with the relativeamount of agent to IAP present in the assay including, for example, 1molar equivalent of agent per IAP, 2 molar equivalents of agent per IAP,5 molar equivalents of agent per IAP, 10 molar equivalents of agent perIAP or 50 molar equivalents of agent per IAP or higher.

An agent that derepresses an IAP-inhibited caspase can also beidentified by its affinity for an IAP or a caspase-binding fragmentthereof, for example, in a binding assay. It will be understood that afunctional fragment of an IAP, caspase or both can be used in a bindingassay to identify a derepressor of an IAP-inhibited caspase. Affinity ofan agent for an IAP determined using a binding assay can, if desired, bequantified by an equilibrium dissociation constant (K_(d)) orequilibrium association constant (K_(a)). An agent that derepresses anIAP-inhibited caspase can be identified as an agent that has a K_(d)that is in the micromolar range including, for example, less than about1×10⁻⁶ M, 1×10⁻⁷ M, or 1×10⁻⁸ M. Higher affinity agents can also beidentified including an agent having nanomolar range affinity such as aK_(d) less than about 1×10⁻⁹ M, 1×10⁻¹⁰ M or 1×10⁻¹¹ M. An agent of theinvention can also have picomolar affinity including, for example, aK_(d) that is less than 1×10⁻¹² M.

Alternatively, the effectiveness of an agent at derepressing anIAP-inhibited caspase can be determined based on inhibition of theassociation between an IAP and caspase, for example, in an inhibitionbinding assay. It will be understood that a functional fragment of anIAP, caspase or both can be used in an inhibition binding assay.Alternatively, a derepressor of an IAP-inhibited caspase can beidentified based on its ability to inhibit binding between IAP andanother inhibitor such as SMAC. An exemplary assay for determininginhibition of IAP binding to SMAC is provided in Example VII. Inhibitioncan be quantified, if desired, by an equilibrium inhibition constant,such as K_(i). Values for K_(i) can be determined by performingderepression assays, such as those described herein, with increasingconcentrations of the agent and a fixed concentration of each bindingpartner. Binding or inhibition can be analyzed to determine theequilibrium constants described above using well known kinetic analysissuch as those described in Segel, Enzyme Kinetics John Wiley and Sons,New York (1975). An agent that derepresses an IAP-inhibited caspase canbe identified as those having K_(i) in the micromolar, nanomolar orpicomolar ranges such as those ranges and values described above forK_(d).

Accordingly, the invention provides a complex having an IAP bound to anagent, the agent having a core peptide or core structure of theinvention including, for example, those core structures described above.The complex can be isolated from at least one other cellular componentnormally occurring with the IAP in nature. For example, the complex canbe in a purified state being substantially free of other cellularcomponents that normally occur with the IAP in nature. The complex canalso occur in a recombinant cell that does not normally express the IAP.

The invention further provides conjugates including a moiety linked toan agent that derepresses an IAP-inhibited caspase. A conjugate of theinvention can include a moiety useful for targeting the agent to aparticular cell or for increasing the stability or biological half lifeof the agent that derepresses an IAP-inhibited caspase. For example, amoiety can be a particular antibody, functional fragment thereof, orother binding polypeptide that has specificity for a particular cell inwhich it is desired to promote apoptosis, such as a tumor cell. Anymoiety capable of targeting the agent to a cell in which anIAP-inhibited caspase is to be derepressed can be used as a conjugate.

A conjugate of an agent that derepresses an IAP-inhibited caspase canalso be a moiety capable of introducing the agent to the cytosol of acell or otherwise facilitating passage of the agent through the cellmembrane. An agent can be introduced into the cell by, for example, aheterologous targeting domain or using a lipid based carrier. Thus, theinvention provides cytosolic delivery of an agent that derepresses anIAP-inhibited caspase.

A moiety can also be a drug delivery vehicle such as a chamberedmicrodevice, a cell, a liposome or a virus that provides stability orproperties otherwise advantageous for administration of the agent thatderepresses an IAP-inhibited caspase. Generally, such microdevices,should be nontoxic and, if desired, biodegradable. Various moieties,including microcapsules, which can contain an agent, and methods forlinking a moiety, including a chambered microdevice, to a therapeuticagent are well known in the art and commercially available (see, forexample, “Remington's Pharmaceutical Sciences” 18th ed. (Mack PublishingCo. 1990), chapters 89-91; Harlow and Lane, Antibodies: A laboratorymanual (Cold Spring Harbor Laboratory Press 1988); see, also, Hermanson,supra, 1996).

In addition, a derepressor of an IAP-inhibited caspase formulation canbe incorporated into biodegradable polymers allowing for sustainedrelease of the compound, the polymers being implanted in the vicinity ofwhere drug delivery is desired, for example, at the site of a tumor orimplanted so that the agent is released systemically over time. Osmoticminipumps also can be used to provide controlled delivery of specificconcentrations of the derepressor of an IAP-inhibited caspase speciesand formulations through cannulae to the site of interest, such asdirectly into a tumor growth or into the vascular supply of a tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991).

A conjugate of the invention can include a moiety that is a label. Alabeled agent that binds to an IAP and/or caspase can be used toidentify the subcellular localization of the IAP and/or caspase or toidentify a previously unidentified IAP or caspase. A labeled agent thatbinds to an IAP and/or caspase can also be used to identify othermolecules that interact with an IAP and/or caspase. As described infurther detail below, such a binding competition assay can be used toidentify an agent that derepresses an IAP-inhibited caspase. A labelthat can be incorporated as a moiety includes, for example, afluorophore, chromophore, paramagnetic spin label, radionuclide, orbinding group having specificity for another molecule that can bedetected.

A labeled agent of the invention can be useful for identifying cellswithin a tissue that are inhibited from apoptosis by an IAP-inhibitedcaspase. Thus, the labeled agent can be used in a diagnostic method toidentify cells for which administration of a derepressor of anIAP-inhibited caspase will allow apoptosis to proceed. The method caninclude steps of administering a labeled agent of the invention to atissue and identifying one or more cells that incorporate the labeledagent. The labeled agent can be administered using methods for in vivodelivery as described above. The diagnostic methods can be used at avariety of resolutions. For example, the method can be carried out toidentify a tissue containing cells labeled by the agent. Alternatively,higher resolution methods can be used to identify a particular cell orcell type within a tissue that is labeled in the presence of anIAP-inhibited caspase. Because the diagnostic methods can be used todistinguish a cell for which administration of a derepressor of anIAP-inhibited caspase will allow apoptosis to proceed from non-labeledcells, the methods can be useful for guiding in the choice of targetingor delivery conjugate to use in a therapeutic method of the invention.

The diagnostic method can be performed in vitro in which case thelabeled agent can be administered by injection or by soaking the tissuein a solution containing the labeled agent. Again the methods can beused at a resolution sufficient to distinguish within a tissue a cellhaving an IAP-inhibited caspase over those that are not inhibited fromapoptosis in this way. Such resolution can be achieved for example, byuse of a microscopic based technique. Further resolution can providesubcellular localization of an IAP-inhibited caspase. Subcellularlocalization can be used to determine an appropriate cytosolic deliveryconjugate or to further identify the role of apoptosis in the particulartissue or cells under study.

The invention also provides a pharmaceutical composition containing aderepressor of an IAP-inhibited caspase and a pharmaceutical carrier.Such compositions can be used in the apoptosis promoting methods of theinvention to inhibit, treat or reduce the severity of a pathologicalcondition characterized by a pathologically reduced level of apoptosis.For example, a derepressor of an IAP-inhibited caspase can beadministered as a solution or suspension together with apharmaceutically acceptable medium.

The derepressor of an IAP-inhibited caspase formulations include thoseapplicable for parenteral administration such as subcutaneous,intraperitoneal, intramuscular, intravenous, intradermal, intracranial,intratracheal, and epidural administration. As well as formulationsapplicable for oral, rectal, ophthalmic (including intravitreal orintracameral), nasal, topical (including buccal and sublingual),intrauterine, or vaginal administration. The derepressor of anIAP-inhibited caspase formulation can be presented in unit dosage formand can be prepared by pharmaceutical techniques well known to thoseskilled in the art. Such techniques include the step of bringing intoassociation the active ingredient and a pharmaceutical carrier orexcipient.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions such as the pharmaceuticallyacceptable media described above. The solutions can additionallycontain, for example, anti-oxidants, buffers, bacteriostats and soluteswhich render the formulation isotonic with the blood of the intendedrecipient. Other formulations include, for example, aqueous andnon-aqueous sterile suspensions which can include suspending agents andthickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and can bestored in a lyophilized condition requiring, for example, the additionof the sterile liquid carrier, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

A pharmaceutically acceptable medium can additionally containphysiologically acceptable compounds that act, for example, to stabilizethe derepressor of an IAP-inhibited caspase agent. Such physiologicallyacceptable compounds include, for example, carbohydrates such asglucose, sucrose or dextrans; antioxidants such as ascorbic acid orglutathione; chelating agents such as EDTA, which disrupts microbialmembranes; divalent metal ions such as calcium or magnesium; lowmolecular weight proteins; lipids or liposomes; or other stabilizers orexcipients. As described previously, derepressor of an IAP-inhibitedcaspase formulation also can be formulated with a pharmaceuticallyacceptable medium such as a biodegradable polymer. All of theabove-described pharmaceutical carriers and media can be what is termedin the art pharmaceutical grade which means that they are of sufficientpurity and quality for use in humans and are distinguishable fromcomparable reagents in research grade formulations.

The invention also provides a composition including a derepressor of anIAP-inhibited caspase and a molecule having therapeutic activity. Amolecule included with a derepressor of the invention can be a compoundhaving activity against a condition characterized by a pathologicallyreduced level of apoptosis. For example, the compound can have activityagainst cancer. An exemplary compound that has activity against prostatecancer and that can be used in combination with a derepressor compoundof the invention is VP-16 (etoposide). As demonstrated by the results ofExample X, administration of VP-16 with either TPI 792-33 or TPI 792-35had a more potent effect on killing cancer cells than any of thesecompounds alone.

Other anti-cancer drugs can also be used in a composition with aderepressor of an IAP-inhibited caspase including, but not limited to,an alkylating agent such as mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide; an antimetabolite such asmethotrexate, 6-mercaptopurine, 5-fluorouracil or cytarabine; anantibody such as Rituxan, Herceptin, or MabThera; a plant alkaloid suchas vinblastine or vincristine, or etoposide; an antibiotic such asdoxorubicin, daunomycin, bleomycin, or mitomycin; a nitrosourea such ascarmustine or lomustine; an inorganic ion such as cisplatin; abiological response modifier such as interferon; an enzyme such asaspariginase; or a hormone such as tamoxifen or flutamide. These andother anti-cancer compounds, including those described herein below withrespect to practicing a therapeutic method of the invention incombination with another therapeutic method, are known in the art andformulations suitable for pharmaceutical use are known as described, forexample, in The Merck Manual 16^(th) Ed., Merck Res. Labs., Rahway N.J.(1992). In addition, for treating a condition characterized by apathologically reduced level of apoptosis, a compound of the inventioncan be administered in conjunction with a therapeutic antibody. Such atherapeutic antibody can be, for example, an antibody that modulatesapoptosis, such as by binding to an apoptosis regulatory molecule andmodulating its activity. As a non-limiting example, a compound of theinvention can be administered in conjunction with an antibody thatactivates caspase 3, caspase 7, Trail-R1 or Trail R-2. ExemplaryTrail-R1 and Trail-R2 monoclonal antibodies are available from HumanGenome Sciences, Rockville, Md.

The invention provides compounds that demonstrate broad anti-canceractivity alone or in combination with known anti-cancer agents. Forexample, polyphenylurea compound of the invention such as TPI 1396-34,TPI 1396-12, TPI 1396-22, and TPI 1396-11 significantly reduce tumorcell growth of sixty different tumor cell lines (see Example XIII andFIGS. 28 and 29). The concentration of polyphenylurea compound requiredto kill 50% of the cells (LD50) was comparable or better than that ofknown anti-cancer drugs. Toxicological analysis of mice treated with TPI1396-12 at dosages effective to inhibit tumor growth indicated no toxiceffects on a variety of parameters including white blood cell count, redblood cell count, platelet count, BUN, bilirubin, ALT and AST (see FIG.41 and Example XXIII). In addition, as shown herein, normal cells wererelatively resistant to polypheylurea compounds compared to tumor celllines (see FIG. 28). Polyphenylurea compounds also were demonstrated toinduce apoptosis in non-replicating malignant cells such as chroniclymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) cellsisolated from patients (see FIG. 28). Additional studies revealed that apolyphenylurea compound of the invention can enhance cytotoxicity ofantigen-specific CTL (see FIG. 39).

As further disclosed herein, polyphenylurea compounds can collaboratewith conventional anticancer drugs to induce killing of tumor cells. Forexample, TPI 1396-34 significantly increases dose-dependent cytoxicityof etoposide (VP16), doxorubicin (DOX) or paclitaxel (TAXOL) in variouscancer cell lines (see Example XIV and FIGS. 30 and 31). Similar effectson the induction of apoptosis were seen using polyphenylurea compoundsand the biological agent TRAIL, which is an apoptosis inducing member ofthe Tumor Necrosis Factor (TNF) family (see FIG. 30).

The invention also provides compounds that demonstrate anti-tumoractivity in clonogenic survival assays and in vivo. For example, thepolyphenylurea compound TPI 1396-34 decreased clonogenic survival ofvarious cancer cell lines in a concentration dependent manner (seeExample XV and FIG. 32). In addition, as disclosed herein,polyphenylurea compounds such as TPI 1396-34 and TPI 1396-22 haveanti-tumor activity in vivo. For example, these compounds significantlyreduced tumor size and tumor weight in human tumor xenografts grown inimmunocompromised mice (see Example XV and FIGS. 32 and 33). Additionalstudies confirmed that the polyphenylurea compounds of the inventionfunction in vivo by modulating caspase activity (see FIG. 40), and thatXIAP protein is indeed the target of these compounds in vivo (see FIG.38).

The invention further provides a kit, including at least one compound ofthe invention that has activity as a derepressor of an IAP-inhibitedcaspase and a second compound having therapeutic activity. A compound ofthe invention that can be included in a kit includes, for example, acompound having a core peptide selected from the group consisting ofCore peptides 4 through 39 and 42 through 55, or having a core structureselected from any of the structures shown in FIGS. 5, 9, 10, 12, 14B,21-24, 34, 35, 36, 37 and 43, wherein the compound derepresses anIAP-inhibited caspase. Such kits are useful, for example, in thetreatment of a condition characterized by a pathologically reduced levelof apoptosis. For example, a kit including VP-16 with either TPI 792-33or TPI 792-35 can be used to treat prostate cancer.

A suitable kit includes compounds as separately packaged formulations orin a mixed formulation, so long as the compounds are provided in anamount sufficient to have a therapeutic effect following at least oneadministration of each compound. The formulations can be any of thosedescribed above, or otherwise known to be appropriate for the particularcompound and mode of administration.

The contents of a kit of the invention are housed in packaging materialor other suitable physical structure, preferably to provide a sterile,contaminant-free environment. In addition, the packaging materialcontains instructions indicating how the materials within the kit can beadministered for treatment of a condition characterized by apathologically reduced level of apoptosis. The instructions for usetypically include a tangible expression describing the route ofadministration or, if required, methods for preparing the formulationfor administration. The instructions can also include identification ofpotential effects from use of the kit's contents or a warning regardingimproper use of the contents of the kit.

The invention provides a method of identifying an agent that derepressesan IAP-inhibited caspase. The method includes the steps of (a)contacting an IAP and a caspase with an agent suspected of being able toderepress an IAP-inhibited caspase, wherein the caspase is anIAP-inhibited caspase that is inhibited by the IAP, wherein thecontacting occurs under conditions that allow caspase activity in theabsence of the IAP; and (b) detecting derepression of the IAP-inhibitedcaspase.

Derepression of the IAP-inhibited caspase can be detected as an increasein an IAP-inhibited caspase activity including, for example, proteolyticactivity.

Proteolytic activity can be measured in an in vitro assay using aspecific substrate. For example, a continuous fluorometric assay can beused to measure hydrolysis rates by following release of either7-amino-4-trifluoromethyl-coumarin (AFC) from DEVD (SEQ ID NO:2) that isderivatized with a C-terminal aminomethylcoumarin, YVAD (SEQ ID NO:3)that is derivatized with a C-terminal aminomethylcoumarin(Tyr-Val-Ala-Asp-aminomethylcoumarin), orcarbobenzoxy-Glu-Val-Asp-aminomethylcoumarin; or by following therelease of p-nitroanilide (pNA) from similar peptides labeled with pNA,as described in U.S. Pat. No. 6,228,603 B1.

An immunoblot or other chromatography based assay can be used to detectproteolysis of a substrate by caspase according to altered molecularweight of the products compared to the substrate. For example, theproteolytic activity of an upstream initiator caspase, such ascaspase-9, can be determined based on processing of a downstreameffector pro-caspase, such as pro-caspase-3, to the mature form in animmunoblot assay as described in U.S. Pat. No. 6,228,603 B1. Comparisonof the results of such an assay for an IAP-inhibited caspase in thepresence and absence of an agent of the invention can be used toidentify a derepressor of the IAP-inhibited caspase according to arelative increase in caspase activity in the presence of the agent.

Proteolytic activity of a caspase can also be determined by identifyingmorphological changes in a cell or a cell nucleus characteristic ofapoptosis. Such changes that are characteristic of apoptosis include,for example, chromatin condensation, nuclear fragmentation, cellshrinkage, or cell blebbing leading to the eventual breakage into smallmembrane surrounded fragments termed apoptotic bodies. Thus, an agentthat is a derepressor of an IAP-inhibited caspase can be identifiedaccording to the ability to cause a characteristic apoptotic change whenadded to a cell that is prevented from undergoing apoptosis by anIAP-inhibited caspase. A similar assay can be performed on a cell freeextract derived from such a cell so long as an apoptotic change such aschromatin condensation or nuclear fragmentation can be distinguished inthe presence and absence of the added agent.

Derepression of an IAP-inhibited caspase can also be detected asdisassociation of an IAP-caspase species. An IAP-inhibited caspase canbe identified as a caspase having an associated IAP using binding assaysknown in the art. Such a complex can be identified according tomolecular weight or size using, for example, non-denaturingpolyacrylamide gel electrophoresis, size exclusion chromatography, oranalytical centrifugation. An IAP-caspase complex can also be identifiedusing a co-precipitation technique. For example, an IAP-caspase complexcan be identified due to the ability of an antibody to co-precipitatewith both partners but not with one or the other partner alone. Similar,techniques can be used when either the IAP or caspase has been modifiedby a recombinant DNA method to incorporate an affinity tag such asglutathione-S-transferase (Amersham Pharmacia; Piscataway, N.J.), whichcan be precipitated with glutathione beads; polyhistidine tag (Qiagen;Chatsworth, Calif.), which can be precipitated with Nickel NTAsepharose; antibody epitopes such as the flag peptide (Sigma; St Louis,Mo.), which can be immunoprecipitated; or other known affinity tag. Anagent that prevents IAP-caspase complex formation or otherwise causesdissociation of the complex can be identified in such an assay as aderepressor of an IAP-inhibited caspase.

The caspases are present in cells as precursor polypeptides referred toas procaspases. Caspase activation occurs due to proteolytic processingof the procaspase. For example, caspase-3 is a heterotetramer composedof approximately 17-20 kDa and 11 kDa polypeptides that are formed byproteolysis of a 32 kDa polypeptide precursor, pro-caspase-3. Cleavageof the pro-caspase-3 proceeds in two steps. The first cleavage resultsin production of a partially processed large subunit (22-24 kDa) thatstill contains the pro-domain, and a smaller, fully processed, subunitof about 11 kDa. In the second step, the pro-domain is cleaved from thepartially processed large subunit, probably by an autocatalytic process,to produce the 17-20 kDa mature, fully processed large subunit of thecaspase-3 enzyme. Removal of the pro-domain, however, is not necessaryfor protease activation, as the partially processed caspase also hascaspase activity.

The methods of the invention for identifying an agent that derepressesan IAP-inhibited caspase can be used to identify a caspase that isprevented from being processed to a mature, fully proteolytically activeform due to the presence of an IAP. For example, the methods can be usedto identify an agent that prevents or suppresses an IAP from inhibitingprocessing of a procaspase to a caspase. Because processing of aprocaspase to a caspase will coincide with an increase in caspaseproteolytic activity, the methods described above for determiningproteolytic activity can be used in a method for identifying an agentthat prevents or suppresses an IAP from inhibiting processing of aprocaspase to a caspase. Similarly, a binding assay, such as thosedescribed above, can be used to identify a procaspase-IAP complexaccording to the combined molecular weight of the partners. An agentthat prevents complex formation or causes the complex to dissociate canbe identified in such an assay as a derepressor of an IAP-inhibitedcaspase. A caspase that is prevented from being processed to a mature,fully proteolytically active form due to the presence of an IAP can alsobe identified according to differences in molecular weight or size ofthe mature and procaspase forms. Thus, an agent that, when contactedwith a procaspase in the presence of an inhibitory IAP, causes a changein molecular weight or size indicative of the mature form can beidentified as a derepressor of an IAP-inhibited caspase.

The methods of the invention can be used to identify a derepressor of anIAP-inhibited caspase that has specificity for a particular IAP orcaspase or combination of a particular IAP and caspase. For example, theinvention provides screening assays for identifying agents that alterthe specific binding of a eukaryotic IAP such as XIAP, c-IAP-1 orc-IAP-2 and a caspase such as caspase-3, caspase-7 or caspase-9. AnyIAP, including any eukaryotic IAP, can be used in a method of theinvention in combination with the appropriate caspase. Other IAPproteins that are involved in regulating particular caspases can beidentified using the methods disclosed herein, then the particularcombination of caspase and IAP can be used in a screening assay toidentify an agent that modulates the regulation of caspase activation bythe IAP or that alters the specific association of the IAP and caspase.

As disclosed herein, invention core peptides were identified byscreening combinatorial libraries having core tetrapeptide andhexapeptide structures. In view of the disclosed methods, the skilledartisan would recognize that combinatorial libraries of peptides havingmore than six amino acids or less than four amino acids also can bescreened to identify other core peptides that derepress an IAP-inhibitedcaspase. Furthermore, while the disclosed methods can be used toinitially identify core peptides that derepress an IAP-inhibitedcaspase, those skilled in the art would know that the methods can beused in an iterative fashion to optimize or to identify additional corepeptides that derepress an IAP-inhibited caspase, as described below.

It is expected that those skilled in the art can use combinatorialsynthetic methods coupled to rapid screening methods to optimize andidentify additional derepressors with increased binding affinity for anIAP or increased activity in derepressing an IAP-inhibited caspase,thereby possessing enhanced therapeutic potential.

The iterative approach is well-known in the art and is set forth, ingeneral, in Houghten et al., Nature, 354, 84-86 (1991); and Dooley etal., Science 266, 2019-2022 (1994); both of which are incorporatedherein by reference. In the iterative approach, for example,sublibraries of a molecule having three variable groups are made whereinthe first variable is defined. Each of the compounds with the definedvariable group is reacted with all of the other possibilities at theother two variable groups. These sub-libraries are each tested to definethe identity of the second variable in the sub-library having thehighest activity in the screen of choice. A new sub-library with thefirst two variable positions defined is reacted again with all the otherpossibilities at the remaining undefined variable position. As before,the identity of the third variable position in the sub-library havingthe highest activity is determined. If more variables exist, thisprocess is repeated for all variables, yielding the compound with eachvariable contributing to the highest desired activity in the screeningprocess. Promising compounds from this process can then be synthesizedon larger scale in traditional single-compound synthetic methods forfurther biological investigation.

The positional-scanning approach has been described for various organiclibraries and for various peptide libraries (see, for example, R.Houghten et al. PCT/US91/08694 and U.S. Pat. No. 5,556,762, both ofwhich are incorporated herein by reference). In the positional scanningapproach sublibraries are made defining only one variable with each setof sublibraries and all possible sublibraries with each single variabledefined (and all other possibilities at all of the other variablepositions) is made and tested. From the instant description one skilledin the art could synthesize libraries wherein 2 fixed positions aredefined at a time. From the testing of each single-variable definedlibrary, the optimum substituent at that position is determined,pointing to the optimum or at least a series of compounds having amaximum of the desired biological activity. Thus, the number ofsublibraries for compounds with a single position defined will be thenumber of different substituents desired at that position, and thenumber of all the compounds in each sublibrary will be the product ofthe number of substituents at each of the other variables.

Phage display methods provide a means for expressing a diversepopulation of random or selectively randomized peptides. Various methodsof phage display and methods for producing diverse populations ofpeptides are well known in the art. For example, Ladner et al. (U.S.Pat. No. 5,223,409, issued Jun. 29, 1993) describe methods for preparingdiverse populations of binding domains on the surface of a phage. Inparticular, Ladner et al. describe phage vectors useful for producing aphage display library, as well as methods for selecting potentialbinding domains and producing randomly or selectively mutated bindingdomains.

An invention derepressor of an IAP-inhibited caspase that containspeptide moieties can be synthesized using amino acids, the active groupsof which are protected as required using, for example, at-butyloxycarbonyl (t-BOC) group or a fluorenylmethoxy carbonyl (FMOC)group. Amino acids and amino acid analogs can be purchased commercially(Sigma Chemical Co., St. Louis Mo.; Advanced Chemtec, Louisville Ky.) orsynthesized using methods known in the art. Peptides synthesized usingthe solid phase method can be attached to a variety of resins,including, for example, 4-methylbenzhydrylamine (MBHA),4-(oxymethyl)-phenylacetamido methyl and4-(hydroxymethyl)phenoxymethyl-copoly(styrene-1% divinylbenzene (Wangresin), all of which are commercially available, or top-nitrobenzophenone oxime polymer (oxime resin), which can besynthesized as described by De Grado and Kaiser, J. Org. Chem. 47:3258(1982).

The choice of amino acids or amino acid analogs incorporated into aninvention peptide will depend, in part, on the specific physical,chemical or biological characteristics required of the derepressor of anIAP-inhibited caspase. Such characteristics are determined by whether,for example, the peptide is to be used in vivo or in vitro, and, whenused in vivo, by the route by which the invention peptide will beadministered or the location in a subject to which it will be directed.For example, the derepressor of IAP-inhibited caspase core peptidesexemplified herein can be synthesized using only L-amino acids. However,the skilled artisan would know that any or all of the amino acids in apeptide of the invention can be a naturally occurring L-amino acid, anon-naturally occurring D-amino acid or an amino acid analog, providedthe peptide can derepress an IAP-inhibited caspase.

The choice of including an L-amino acid or a D-amino acid in theinvention peptides depends, in part, on the desired characteristics ofthe peptide. For example, the incorporation of one or more D-amino acidscan confer increased stability on the peptide in vitro or in vivo. Theincorporation of one or more D-amino acids also can increase or decreasethe activity, such as IAP binding affinity, of the peptide asdetermined, for example, using the assay described herein in Example VIIor other well known methods for determining the binding affinity of aparticular peptide to a particular protein.

As set forth above, invention peptides can be either linear, cyclic ormultivalent, and the like, which conformations can be achieved usingmethods well-known in the art. As used herein a “cyclic” peptide refersto analogs of synthetic linear peptides that can be made by chemicallyconverting the structures to cyclic forms. Cyclization of linearpeptides can modulate bioactivity by increasing or decreasing thepotency of binding to the target protein (Pelton, J. T., et al., Proc.Natl. Acad. Sci., U.S.A., 82:236-239). Linear peptides are very flexibleand tend to adopt many different conformations in solution. Cyclizationacts to constrain the number of conformations available in solution, andthus, can favor a conformation having a higher affinity for IAP or morepotent activity as a derepressor of an IAP-inhibited caspase.

Cyclization of linear peptides is accomplished either by forming apeptide bond between the free N-terminal and C-terminal ends (homodeticcyclopeptides) or by forming a new covalent bond between amino acidbackbone and/or side chain groups located near the N- or C-terminal ends(heterodetic cyclopeptides) (Bodanszky, N., 1984, supra). The lattercyclizations use alternate chemical strategies to form covalent bonds,e.g. disulfides, lactones, ethers, or thioethers. Linear peptides offive or more amino acid residues, as described herein, can be cyclizedrelatively easily. The propensity of the peptide to form a beta-turnconformation in the central four residues facilitates the formation ofboth homo- and heterodetic cyclopeptides. The presence of proline orglycine residues at the N- or C-terminal ends also facilitates theformation of cyclopeptides, especially from linear peptides shorter thansix residues in length.

An agent of the invention can be multivalent with respect to the numberof derepressor IAP-inhibited caspase sequences or moieties are presentper molecule. The sequences or moieties present in a multivalent agentcan be either the same or different. Exemplary multivalent peptides canbe produced using the well-known multiple antigen peptide system (MAPS;see, e.g., Briand et al., 1992, J. Immunol Meth., 156(2):255-265; Schottet al., 1996, Cell Immun., 174(2):199-209, and the like). An agent thatis multivalent with respect to the number of derepressor IAP-inhibitedcaspase sequences or moieties present can be useful for interacting withan IAP having more than one BIR domain. For example, a single agent canbe made to contain two or more sequences or moieties that interact withseparate BIR domains on the same IAP. The presence of multipleinteracting partners in the multivalent agent and IAP can increaseaffinity or specificity of the interaction.

In some cases, it can be desirable to allow a derepressor of anIAP-inhibited caspase to remain active for only a short period of time.In those cases, the incorporation of one or more L-amino acids in theagent can allow, for example, endogenous peptidases in a subject todigest the agent in vivo, thereby limiting the subject's exposure to thederepressor. In one embodiment, the agent, whether based on a peptidebackbone or other structure, can include a peptide linkage through anL-aspartate moiety or residue. Degradation of the L-aspartate containingagent by the caspases that it derepresses can provide a feedback controlmechanism minimizing the extent of apoptosis allowed by the agent. Theskilled artisan can determine the desirable characteristics required ofan invention agent by taking into consideration, for example, the ageand general health of a subject, and the like. The half life in asubject of a peptide having, for example, one or more D-amino acidssubstituted for a corresponding L-amino acid can be determined usingmethods well known to those in the field of pharmacology.

Selective modification of the reactive groups in a peptide also canimpart desirable characteristics to a derepressor of an IAP-inhibitedcaspase. An invention peptide can be manipulated while still attached tothe resin to obtain, for example, an N-terminal modified peptide such asan N-acetylated peptide. Alternatively, the peptide can be removed fromthe resin using hydrogen fluoride or an equivalent cleaving reagent andthen modified. Agents synthesized containing the C-terminal carboxygroup (Wang resin) can be modified after cleavage from the resin or, insome cases, prior to solution phase synthesis. Methods for modifying theN-terminus or C-terminus of a peptide are well known in the art andinclude, for example, methods for acetylation of the N-terminus andmethods for amidation of the C-terminus.

Also encompassed within the scope of invention peptides are peptideanalogs. As used herein, the term “peptide analog” includes any peptidehaving an amino acid sequence substantially the same as a sequencespecifically shown herein, such as Core peptides 1 through 55, in whichone or more residues have been conservatively substituted with afunctionally similar residue and which displays the ability tofunctionally mimic an invention lectin-binding peptide as describedherein. Examples of conservative substitutions include the substitutionof one non-polar (hydrophobic) residue such as isoleucine, valine,leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

As used herein the phrase “conservative substitution” also includes theuse of a chemically derivatized residue in place of a non-derivatizedresidue, provided that such peptide displays the required IAP binding orinhibiting activity. A chemical derivative can include, for example,those molecules in which free amino groups have been derivatized to formamine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and omithine may be substituted for lysine.Peptides of the present invention also include any peptide having one ormore additions, deletions or combination of additions and deletions ofresidues, relative to the sequence of a peptide whose sequence is shownherein, so long as the required IAP binding or inhibiting activity ismaintained.

Those skilled in the art will recognize from the guidance providedherein that an agent of the invention can include a core peptide or corestructure that is modified, derivatized, or substituted with an analogsor derivative so long as the agent is capable of derepressing anIAP-inhibited caspase. Such alterations in a core peptide or corestructure can be made by well known synthetic methods such as thosedescribed herein. An agent so altered can be tested for activity usingthe methods described herein such as the derepression assay described inExample I or the polarization binding assay described in Example VII.

An agent identified as a derepressor of an IAP-inhibited caspase can betested using an assay for determining caspase proteolytic activity orbinding of IAP and caspase in the presence or absence of the agentincluding, for example, the assays described above. An agent that isidentified as capable of derepressing an IAP-inhibited caspase usingsuch assays can be further combinatorialized at one or more positionsusing the iteration approach described above. Alternatively, anidentified derepressor or plurality of derepressors can be used as abasis for the rational design of second generation agents. For example,common structural features between a plurality of validated agents canbe used to guide the synthesis of a generalized structure incorporatingthose shared features. Structural information regarding an agent whenbound to an IAP or caspase can also be used to design a secondgeneration agent that retains or improves upon moieties identified asproviding favorable interactions while removing moieties that lead tounfavorable interactions with the caspase or IAP.

The invention provides structure activity relationship (SAR) informationof polyphenylurea compounds which is used in designing optimized secondgeneration agents (see Example XVI and Table X and XI). A series ofcompounds, shown in FIG. 34 (TPI 1509), were synthesized based on theTPI 1396 polyphenylurea compounds. All of these compounds were active inthe XIAP derepression assay. Therefore, the invention provides anisolated agent having a core structure selected from any of thestructures shown in FIG. 34 where the agent derepresses an IAP-inhibitedcaspase. In addition, modifications of R groups of a compound from theTPI 1509 series are provided herein in Example XVII (see also FIG. 35).As understood by one skilled in the art, these types of R groupmodifications can be used for other polyphenylurea compounds such asthose in the TPI 1396 library.

The invention provides a method of identifying an agent that derepressesan IAP-inhibited caspase. The method includes the steps of (a) detectinga labeled derepressor of an IAP-inhibited caspase bound to an IAP orcaspase; (b) contacting the bound IAP or caspase with a candidate agent,the candidate agent suspected of being able to derepress anIAP-inhibited caspase; and (c) detecting dissociation of the labeledderepressor of an IAP-inhibited caspase from the IAP or caspase, wherebythe candidate agent is identified as an agent that derepresses anIAP-inhibited caspase. A labeled derepressor of an IAP-inhibited caspaseused in the method can have a core motif selected from a core peptide ofthe invention such as Core peptides 4 through 39 and 42 through 55 or acore structure selected from TPI 759, TPI 882, TPI 914, TPI 927, or acompound having a structure selected from TPI 1391, TPI 1349, TPI 1400,TPI 1396, TPI 1509, TPI 1540, TPI 1577, TPI 1567, TPI 1572, TPI 792 andTPI 1332. The methods can be used to identify a better derepressor of anIAP-inhibited caspase in a screening format as described above and inthe Examples.

The invention also provides a novel negative regulatory binding site onan IAP. The novel negative regulatory binding site identified hereindoes not bind to the SMAC peptide (also known as DIABLO). The SMACpeptide is known to bind to IAPs such as XIAP through the BIR3 domain ofthe IAP (Liu et al, Nature 408:1004-1008 (2000)). As disclosed herein inFIG. 26, an active polyphenylurea compound such as TPI 1396-34 does notcompete with biotin-SMAC 7-mer peptide AVPIAQK (SEQ ID NO: 5) forbinding to XIAP. As further disclosed herein in Example XVII and FIG.36, an active tetrapeptide compound such as TPI 1332-69 or TPI 1332-4does not compete with biotin-SMAC 7-mer peptide AVPIAQK (SEQ ID NO: 5)for binding to XIAP. Therefore, disclosed herein are compounds that actthrough a non-SMAC binding site on an IAP such as XIAP. As is furtherdisclosed herein, the non-SMAC binding site of XIAP has been identifiedto be the BIR2 domain. The ability of polyphenylurea compounds of theinvention to derepress XIAP BIR2 domain-inhibited caspase is disclosedherein, for example, in FIGS. 21C, 22E, 23E and 24G In addition, theability of polyphenylurea compounds of the invention to bind directly toBIR2 is shown in FIG. 42 and described in Example XXIV.

These results indicate that agent that derepresses an IAP-inhibitedcaspase can function by binding to a BIR domain of the IAP and therebyreducing the ability of the BIR domain to block caspase-IAP function.Therefore, the invention provides a method of identifying an agent thatderepresses an XIAP-inhibited caspase that involves (a) contacting acaspase and an BIR domain, wherein the BIR domain is capable ofinhibiting the caspase, under conditions that allow caspase activity inthe absence of the BIR domain, with a candidate agent, and (b) detectingcaspase activity, wherein an increase in the activity of the inhibitedcaspase identifies an agent that derepresses an IAP-inhibited caspase.

The method of the invention for identifying an agent that derepresses anIAP-inhibited caspase involves contacting a caspase with a BIR domainthat is capable of inhibiting the caspase. Any BIR domain that iscapable of inhibiting a caspase can be used in the methods of theinvention. The ability of a BIR domain to inhibit caspase activity canbe determined using a variety of well known methods, for example, bydetermining a lower level of hydrolysis of a specific substrate by thecaspase in the presence of the BIR domain as compared to the activity inthe absence of the BIR domain. Given the role of caspases in apoptosis,it will be recognized by those skilled in the art that caspase activitycan be identified directly, for example, by examining proteolysis(hydrolysis) of a specific substrate or indirectly, for example, byidentifying morphological changes in a cell or cell nucleuscharacteristic of apoptosis. Exemplary assays for detecting caspaseactivity are described herein above and in Example I. An example of aBIR domain capable of inhibiting caspase-3 is the BIR2 domain of XIAP(see Example VIII).

The invention further provides a method of identifying an agent thatderepresses an IAP-inhibited caspase by (a) detecting a labeledderepressor of an IAP-inhibited caspase bound to a non-SMAC binding siteon the IAP; (b) contacting the bound IAP or caspase with a candidateagent, the candidate agent suspected of being able to derepress anIAP-inhibited caspase; and (c) detecting dissociation of the labeledderepressor of an IAP-inhibited caspase from the IAP or caspase, wherebythe candidate agent is identified as an agent that derepresses anIAP-inhibited caspase. In one embodiment, the labeled derepressor isbased on a core structure from the TPI 1332 or TPI 1396 library. Inanother embodiment, the non-SMAC binding site on the IAP is a site boundby TPI 1332-69 or TPI 1332-4.

In a further embodiment, the non-SMAC binding the on IAP is a BIRdomain. Therefore, the method can be practiced by (a) detecting alabeled derepressor of a BIR domain-inhibited caspase, the derepressorbound to the BIR domain of a BIR domain-caspase complex; (b) contactingthe BIR domain-caspase complex with a candidate agent, the candidateagent suspected of being able to derepress a BIR domain-inhibitedcaspase, and (c) detecting dissociation of the labeled derepressor ofthe BIR domain-inhibited caspase from the complex, wherein thederepressor is selected from an isolated agent comprising a corestructure selected from TPI 1391, TPI 1349, TPI 1396, TPI 1509, TPI1540, TPI 1400, TPI 792 and TPI 1332, whereby the candidate agent isidentified as an agent that derepresses an IAP-inhibited caspase.

As is described in Example XXIV, a compound of the invention thatderepresses an XIAP-inhibited caspases can bind directly to the BIR2domain of XIAP. It is recognized that an agent capable of competing witha compound of the invention that binds to a BIR2 domain will also bindto the BIR2 domain at a site important for derepression activity.Therefore, the invention provides a method for identifying an agent thatderepresses an IAP-inhibited caspase based on the ability of the agentto compete with a compound of the invention for binding to a BIR2domain. The method involves (a) contacting a BIR2 domain with acandidate agent in the presence of a derepressor of an IAP-inhibitedcaspase, under conditions wherein the BIR2 domain binds to thederepressor, and (b) detecting dissociation of the derepressor from theBIR2 domain, whereby the candidate agent is identified as an agent thatderepresses an IAP-inhibited caspase, wherein the derepressor isselected from an isolated agent comprising a core structure selectedfrom TPI 1391, TPI 1349, TPI 1396, TPI 1509, TPI 1540, TPI 1400, TPI 792and TPI 1332.

A variety of assays are well known in the art that can be used toidentify an agent that derepresses an IAP-inhibited caspase. Suchmethods include binding assays where candidate agents are added to acomplex that contains a derepressor and an IAP such as XIAP. Thederepressor or IAP can be immobilized, for example to a latex bead orplate or can be free in solution. The derepressor, IAP or candidateagent can be conjugated to a radiolabel, fluorescent label or enzymelabel such as alkaline phosphatase, horse radish peroxidase orluciferase. For example, a candidate agent can be added to a complexwhich contains an IAP and a labeled derepressor, for example, where theIAP is immobilized on a solid support such as a latex bead. The amountof labeled derepressor that is displaced by the candidate agent can thenbe determined. Alternatively, this assay can be performed where the IAPis not bound to a solid support but is free in solution. In addition,fluorescently labeled candidate compounds can also be added to a complexthat contains a derepressor and IAP and bound complexes that contain thelabeled candidate agent can be detected, for example, using afluorescence polarization assay (Degterev et al., Nature Cell Biology3:173-182 (2001)).

One skilled in the art understands that a variety of additional meanscan be used to determine whether a candidate agent is an agent thatderepresses an IAP-inhibited caspase or whether the candidate agent candisplace a derepressor bound to an IAP. For example, a scintillationproximity assay (Alouani, Methods Mol. Biol. 138:135-41 (2000)) can beused. Scintillation proximity assays involve the use of afluomicrosphere coated with an acceptor molecule, such as an antibody,to which an antigen will bind selectively in a reversible manner. Forexample, an IAP-derepressor complex can be bound to a fluomicrosphereusing an antibody that specifically binds to the IAP, and contacted witha ³H or ¹²⁵I labeled FP candidate agent. If the labeled candidate agentspecifically binds to the IAP, the radiation energy from the labeledcandidate agent is absorbed by the fluomicrosphere, thereby producinglight which is easily measured.

Additional assays suitable for identifying an agent that derepresses anIAP-inhibited caspase and for determining specific binding of acandidate agent to an XIAP after displacing a derepressor can include,without limitation, UV or chemical cross-linking assays (Fancy, Curr.Opin. Chem. Biol. 4:28-33 (2000)) and biomolecular interaction analyses(Weinberger et al., Pharmacogenomics 1:395-416 (2000)). Specific bindingof a candidate agent to an IAP can be determined by cross-linking thesetwo components, if they are in contact with each other, using UV or achemical cross-linking agent. In addition, a biomolecular interactionanalysis (BIA) can detect whether two components are in contact witheach other. In such an assay, one component, such as an IAP-derepressorcomplex is bound to a BIA chip, and a second component such as acandidate agent is passed over the chip. If the candidate agentdisplaces the derepressor and binds to the IAP, the contact results inan electrical signal, which is readily detected.

Further assays suitable for identifying an agent that derepresses anIAP-inhibited caspase include those based on NMR methods. Such methodstake advantage of the significant perturbations that can be observed inNMR-sensitive parameters of a candidate agent or its target, such as anIAP or domain thereof, that occur upon complex formation between theagent and target. These perturbations can be used to detect bindingbetween a candidate agent and IAP, as well as to assess the strength ofthe binding interaction. In addition, some NMR techniques allow theidentification of the agent binding site or which part of the agent isresponsible for interacting with the target. Exemplary NMR methodsuseful for identifying an agent that derepresses an IAP-inhibitedcaspase include “SAR by NMR,” which is described, for example, in Shukeret al. Science, 274, 1531-1534 (1996), and a variety of NMR-basedscreening assays, including SHAPES screening, fragment-based approachesfor lead optimization using NMR, and fluorine-NMR competition bindingexperiments, all of which are described, for example, in CombinatorialChemistry & High Throughput Screening, Vol. 5, No. 8 (2002) and inHajduk et al., Quarterly Reviews of Biophysics 32(3):211-240 (1999).

Fluorescence-based assays are also suitable for identifying an agentthat derepresses an IAP-inhibited caspase. Examples of fluorescencemethods applicable to determining an interaction between an agent thatderepresses an IAP-inhibited caspase and its corresponding target, suchas an IAP or caspase, include observations fluorescence intensitychanges resulting from an alteration in interaction between agent andtarget; fluorescence resonance energy transfer (FRET), which is usefulfor determining change in fluorescence intensity based on distancebetween agent and target; fluorescence polarization changes resulting achange in size of an observed binding partner when associated ordissociated from the another binding partner; fluorescence lifetimechanges, and fluorescence correlation spectroscopy, which is based ontranslation diffusion, a parameter related to the size of an observedbinding partner. Such methods can involve employing a fluorescentlylabeled agent or binding partner. For example, a fluorophore can bedetected based on the excitation or emission wavelengths of thefluorophore, fluorescence polarization of the fluorophore, or intensityof fluorescence emitted from the fluorophore. Alternatively, detectioncan be based on a difference in a measurable property of the label forthe bound and unbound state. For example, as demonstrated in ExampleVII, difference in fluorescence polarization due to the slower rotationof a substrate bound to an IAP compared to the unbound substrate can beused to detect association. Other measurable differences that can beused to determine association of a fluorophore-labeled agent with an IAPor caspase include, for example, different emission intensity due to thepresence or absence of a quenching agent, difference in emissionwavelength due to the presence or absence of a fluorescence resonanceenergy transfer (FRET) donor or acceptor, or difference in emissionwavelength due to differences in fluorophore conformation orenvironment. A derepressor of an IAP-inhibited caspase used in a methodof the invention can be labeled with any of a variety of labelsincluding, for example, those described above. A labeled derepressorthat is bound to an IAP or caspase can be detected according to a knownmeasurable property of the label.

Dissociation of the labeled derepressor of an IAP-inhibited caspase fromthe IAP or caspase can be detected as absence or reduction in the amountof label from the IAP or caspase in the presence of a competitivebinding candidate agent or as a reversal of a change that occurs uponassociation of the labeled agent with a caspase or IAP in the presenceof a competitive binding candidate agent. Thus, dissociation can bedetected in the presence of a non-labeled candidate agent as a reductionor loss of radioactivity of the IAP or caspase in the presence of aradionuclide labeled derepressor, reduction or loss of electromagneticabsorbance at a specified wavelength for the IAP or caspase in thepresence of a chromophore labeled derepressor, reduction or loss ofmagnetic signal at a specified field strength or radio frequency for theIAP or caspase in the presence of a paramagnetic spin labeledderepressor or reduction or loss of a secondary label associated withthe IAP or caspase in the presence of a derepressor that is labeled witha binding group for the secondary label. An example of dissociationmeasured by the reversal of a change occurring upon association isprovided in Example VII, where a difference in polarization due to thefaster rotation of a dissociated substrate compared to the IAP-boundsubstrate is used to detect dissociation.

Other changes in a property of a label that can be detected to determineassociation or dissociation of an appropriately labeled derepressor andIAP or caspase include, for example, absorption and emission of heat,absorption and emission of electromagnetic radiation, affinity for areceptor, molecular weight, density, mass, electric charge,conductivity, magnetic moment of nuclei, spin state of electrons,polarity, molecular shape, or molecular size. Properties of thesurrounding environment that can change upon association or dissociationof an appropriately labeled derepressor and IAP or caspase include, forexample, temperature and refractive index of surrounding solvent.Association and dissociation of a derepressor from an IAP or caspase canbe measured based on any of a variety of properties of a labeledderepressor or of the complex between a derepressor and IAP or caspaseusing well known methods including, for example, equilibrium bindinganalysis, competition assays, and kinetic assays as described in Segel,Enzyme Kinetics John Wiley and Sons, New York (1975), and Kyte,Mechanism in Protein Chemistry Garland Pub. (1995).

In addition, virtual computational methods and the like can be used toidentify compounds that can displace a derepressor in a screening methodof the invention. Exemplary virtual computational methodology involvesvirtual docking of small-molecule agents on a virtual representation ofan IAP or IAP-derepressor complex structure in order to determine orpredict specific binding. See, for example, Shukur et al., supra, 1996;Lengauer et al., Current Opinions in Structural Biology 6:402-406(1996); Choichet et al., Journal of Molecular Biology 221:327-346(1991); Cherfils et al., Proteins 11:271-280 (1991); Palma et al.,Proteins 39:372-384 (2000); Eckert et al., Cell 99:103-115 (1999); Looet al., Med. Res. Rev. 19:307-319 (1999); Kramer et al., J. Biol. Chem.(2000).

The methods of the invention for identifying an agent that derepressesan IAP-inhibited caspase can be performed using low throughput or highthroughput assay formats. Screening can be carried out in all plateformats, including for example, 96, 384 and 1536 well formats. Inaddition, assays such as those described above can be performed inkinetic-based or end point-based formats. To increase screeningthroughout, more than one candidate agent or caspase can be present inan assay sample. The number of different candidate agents to test in themethods of the invention will depend on the application of the method.For example, one or a small number of candidate agents can be screenedusing manual screening procedures, or when it is desired to compareefficacy among several candidate agents. However, it will be appreciatethat the larger the number of candidate agents, the greater thelikelihood of identifying a n agent having the desired activity in ascreening assay. Additional, large numbers of candidate agents can beprocessed in high-throughput automated screening methods.

The invention further provides a method for identifying a derepressor ofan IAP-inhibited caspase in a database. A database of molecules such aspeptides or small molecules can be queried with the structure of aderepressor of an IAP-inhibited caspase to identify candidate agentshaving a moiety identical or similar to the query structure. A candidateagent identified in a database search can be synthesized, isolated orotherwise obtained using known methods and then tested for its level ofactivity as a derepressor of an IAP-inhibited caspase using the assaysdescribed above and in the Examples.

For peptide based derepressors, a query can be made to a database basedon amino acid sequence (primary structure) or three dimensionalstructure (tertiary structure) or a combination of both to identifypeptides or proteins having identical or substantially similarstructures. Methods for comparing primary sequence structure which canbe used to determine that two sequences are substantially the same arewell known in the art as are databases including, for example, SwissProtand GenPept. For example, one method for determining if two sequencesare substantially the same is BLAST, Basic Local Alignment Search Tool,which can be used according to default parameters as described byTatiana et al., FEMS Microbial Lett. 174:247-250 (1999) or on theNational Center for Biotechnology Information web page. BLAST is a setof similarity search programs designed to examine all available sequencedatabases and can function to search for similarities in amino acid ornucleic acid sequences. A BLAST search provides search scores that havea well-defined statistical interpretation. Furthermore, BLAST uses aheuristic algorithm that seeks local alignments and is therefore able todetect relationships among sequences which share only isolated regionsof similarity including, for example, protein domains (Altschul et al.,J. Mol. Biol. 215:403-410 (1990)).

In addition to the originally described BLAST (Altschul et al., supra,1990), modifications to the algorithm have been made (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). One modification is GappedBLAST, which allows gaps, either insertions or deletions, to beintroduced into alignments. Allowing gaps in alignments tends to reflectbiologic relationships more closely. For example, gapped BLAST can beused to identify sequence identity within similar domains of two or morepolypeptides. A second modification is PSI-BLAST, which is a sensitiveway to search for sequence homologs. PSI-BLAST performs an initialGapped BLAST search and uses information from any significant alignmentsto construct a position-specific score matrix, which replaces the querysequence for the next round of database searching. A PSI-BLAST search isoften more sensitive to weak but biologically relevant sequencesimilarities.

A second resource that can be used to determine if two sequences aresubstantially the same is PROSITE, available on the world wide web atExPASy. PROSITE is a method of determining the function ofuncharacterized polypeptides translated from genomic or cDNA sequences(Bairoch et al., Nucleic Acids Res. 25:217-221 (1997)). PROSITE consistsof a database of biologically significant sites and patterns that can beused to identify which known family of polypeptides, if any, the newsequence belongs. Using this or a similar algorithm, a polypeptide thatis substantially the same as another polypeptide can be identified bythe occurrence in its sequence of a particular cluster of amino acidresidues, which can be called a pattern, motif, signature orfingerprint, that is substantially the same as a particular cluster ofamino acid residues in a reference polypeptide including, for example,those found in similar domains. PROSITE uses a computer algorithm tosearch for motifs that identify polypeptides as family members. PROSITEalso maintains a compilation of previously identified motifs, which canbe used to determine if a newly identified polypeptide is a member of aknown family.

Tertiary structure of a derepressor of an IAP-inhibited caspase can bedetermined by a theoretical method such as ab initio protein foldingusing algorithms known in the art or by an empirical method such asX-ray crystallographic or nuclear magnetic resonance based structuredetermination. A structural model of a derepressor can be used in analgorithm that compares polypeptide structure including, for example,SCOP, CATH, or FSSP which are reviewed in Hadley and Jones, Structure7:1099-1112 (1999) and regions having a particular fold or conformationused as a region for sequence comparison to a second polypeptide toidentify substantially similar regions.

Similar database searching methods can be used for non-peptide basedderepressors or to query a database of non-peptide based candidateagents based on structure. A database can be searched, for example, byquerying based on chemical property information or on structuralinformation. In the latter approach, an algorithm based on finding amatch to a template can be used as described, for example, in Martin,“Database Searching in Drug Design,” J. Med. Chem. 35:2145-2154 (1992).

A derepressor of an IAP-inhibited caspase can also be identified in adatabase using the results of a positional scanning syntheticcombinatorial library as a query. Such results can be represented as amotif and the motif used to search a database for a derepressor of anIAP-inhibited caspase. Motif searches are generated from screeningresults of positional scanning synthetic combinatorial libraries, andcontained in each position are amino acids corresponding to mixtureshaving an activity threshold greater than a specified value. An exampleof an activity threshold is the ratio of V_(max) for caspase activity inthe presence and absence of a candidate agent as described in Example I.Motif based database searching is known in the art as described, forexample, in Hemmer et al., Nat. Med. 5:1375-1382 (1999), Hemmer et al.,J. Exp. Med. 185:1651-1659 (1997) and Hemmer et al., Immunol Today19:163-168 (1998).

Alternatively, results from a positional scanning syntheticcombinatorial library can be represented as a score matrix and the scorematrix used to query for other derepressors of an IAP-inhibited caspasein a sequence database. Methods for identifying candidate peptides orproteins based on score-matrix based searches of a databases aredescribed in Zhao et al., J. Immunol. 167:2130-2141 (2001). Briefly, amatrix is constructed in which columns represent positions, rowsrepresent the 20 amino acids and each is correlated with a score. Thescore for a particular position and amino acid is based on assay resultsfor the mixture of a positional scanning synthetic combinatorial librarycorresponding to that amino acid defined at that position. For example,each score can correspond to the ratio of V_(max) for caspase activityin the presence and absence of the mixture corresponding to the aminoacid defined at the particular position. The scoring matrix is then usedto search for candidate derepressors of an IAP-inhibited caspase bymoving the scoring matrix across database entries in 1 amino acidincrements. A score is calculated for the database entries searched andeach is ranked. Those having a score above a predetermined cutoff areidentified as candidate derepressors of an IAP-inhibited caspase.

The invention provides a method of derepressing an IAP-inhibitedcaspase, by contacting an IAP-inhibited caspase with an effective amountof an agent to derepress an IAP-inhibited caspase, the agent having acore motif selected from a core peptide of the invention, such as corepeptides 4 through 39 and 42 through 55, or a core structure of theinvention such as TPI 759, TPI 882, TPI 914 or TPI 927.

For inhibiting a caspase inhibitory activity of an inhibitor ofapoptosis protein (IAP), the IAP-inhibited caspase is contacted with anamount of derepressor effective to derepress the IAP-inhibited caspase.Thus, an effective amount of the agent is an amount that is sufficientto yield an increase in caspase proteolytic activity from thederepressed IAP-inhibited caspase compared to the caspase activity foran IAP-inhibited caspase. An increase in proteolytic activity from aderepressed IAP-inhibited caspase can be determined using any of themethods described above in reference to a method for identifying aderepressor of an IAP-inhibited caspase.

An agent of the invention can be contacted with an IAP-inhibited caspaseunder conditions suitable for caspase activity to occur once an IAP isinhibited from inhibiting the caspase. Such conditions include thosedescribed in Example I. The agent that is contacted with theIAP-inhibited caspase can be present in a mixture of compounds, in anisolated form or in substantially pure form. As described above, amixture of compounds can be contacted with an IAP-inhibited caspase in ascreening method employing positional scanning or iteration. Such amixture can be identified as having the ability to derepress anIAP-inhibited caspase. The mixture can be used in the methods of theinvention to derepress an IAP inhibited caspase. Alternatively, aparticular species in the mixture having such activity can be furtherdefined by isolating individual species in the mixture and repeating thederepression assay or performing a second assay for derepression of anIAP-inhibited caspase. An agent that derepresses an IAP-inhibitedcaspase can be contacted with the IAP-inhibited caspase in asubstantially pure form, as a conjugate or in a formulation as describedabove.

In a further embodiment of the invention an IAP-inhibited caspase can becontacted with an agent of the invention in a cell. Accordingly, theinvention provides a method of promoting apoptosis in a cell, bycontacting the cell with an effective amount of an agent to derepress anIAP-inhibited caspase, the agent having a core motif selected from acore peptide of the invention, such as Core peptides 4 through 39 and 42through 55, or a core structure of the invention such as TPI 759, TPI882, TPI 914 or TPI 927.

Methods described herein for cytosolic delivery of an IAP-inhibitedcaspase, such as attachment of a moiety of conjugate, can be used in amethod of promoting apoptosis in a cell. An effective amount of theagent can be identified as an amount sufficient to allow apoptosis tooccur in the cell. Methods of determining morphological changes in acell or nucleus that are characteristic of apoptosis, such as thosedescribed above in relation to identifying a derepressor of anIAP-inhibited caspase, can be used to monitor apoptosis while performinga method of promoting apoptosis in a cell.

The invention also provides a method for reducing the ability of apopulation of cells to survive ex vivo. The method can include the stepsof contacting the cells with an agent of the invention, wherein theagent derepresses an IAP-inhibited caspase. The cells can be contactedwith the agent using the methods described above for promoting apoptosisin a cell. The methods can be used to remove a particular subpopulationof cells in a sample using the targeting methods described above, suchas the attachment of a targeting moiety to the agent.

The methods of the invention can be carried out in a cell from anyorganism in which apoptosis can occur when an IAP-inhibited caspase isderepressed including, for example, a eukaryotic cell, such as amammalian cell, human cell, non human-primate cell, mouse cell, hamstercell, or other animal cell; an invertebrate cell such as a fly ornematode cell or a yeast cell. Various cell types can be used in themethods of the invention including, for example, a tumor cell, stemcell, neural cell, fat cell, hematopoietic cell, liver cell or musclecell. In particular the methods are useful for inducing apoptosis inaberrantly regulated cells including, for example, cells that exhibituncontrolled cell proliferation as well as cells that exhibitdysfunction in specific phases of the cell cycle, leading to alteredproliferative characteristics or morphological phenotypes. Specificexamples of aberrantly regulated cell types include neoplastic cellssuch as cancer and hyperplastic cells characteristic of tissuehyperplasia. Another specific example includes immune cells that becomeaberrantly activated or fail to down regulate following stimulation.Autoimmune diseases are mediated by such aberrantly regulated immunecells. Aberrantly regulated cells also include cells that arebiochemically or physiologically dysfunctional. Other types of aberrantregulation of cell function or proliferation are known to those skilledin the art and are similarly target cells of the invention applicablefor apoptotic destruction using the methods of the invention.

Because a number of characteristic changes associated with apoptosis ofa cell are due to the proteolytic activity of caspases, the methods canbe used to induce characteristic changes of apoptosis. For example,caspase induced proteolysis of lamin B, which is involved in attachmentof chromatin to the nuclear envelope, can be responsible for collapse ofthe chromatin associated with apoptosis (Martin and Green, supra, 1995).Caspase induced proteolysis of the 45 kDa subunit of DNA fragmentationfactor (DFF-45) activates a pathway leading to fragmentation of genomicDNA into nucleosomal fragments (Liu et al., Cell 89:175-184 (1997)). Inaddition, caspase induced proteolysis of PARP can prevent the ability ofPARP to repair DNA damage, further contributing to the morphologicchanges associated with apoptosis. Thus, the methods of the inventioncan be used to induce collapse of the chromatin and fragmentation ofgenomic DNA associated with apoptosis. Other caspase target proteinsinclude sterol regulatory element binding proteins; retinoblastoma (RB)protein; DNA-dependent kinase; U1 70-K kinase; and the large subunit ofthe DNA replication complex (Wang et al., EMBO J. 15:1012-1020 (1996);Takahashi et al., Proc. Natl. Acad. Sci. USA 93:8395-8400 (1996);Casciola-Rosen et al., J. Exp. Med. 183:1957-1964 (1996); and Ubeda andHabener, J. Biol. Chem. 272:19562-19568 (1997)) each of which can beinduced to be proteolyzed by the methods of the invention.

In mammalian cells, activation of caspases is achieved through at leasttwo independent mechanisms, which are initiated by distinct caspases butresult in activation of common “executioner” caspases. Apoptosisinitiated by ligand binding to the Fas receptor is one well describedcell death pathway. In this pathway, binding of a ligand to Fas allowsthe intracellular domain of Fas to bind the intracellular MORT1 (FADD)protein, which, in turn, binds to caspase-8 (MACH; FLICE; Mch5; seeBoldin et al., Cell 85:803-815 (1996); Muzio et al., Cell 85:817-827(1996)). These results define caspase-8 as an upstream caspase involvedin the Fas cell death pathway. In addition, caspase-3 is activated inthe Fas cell death pathway, suggesting that an upstream protease such ascaspase-8 or a protease activated by caspase-8 is involved in caspase-3activation. Accordingly, the methods of the invention can be used todirectly derepress IAP inhibited-caspase-8 thereby effectivelyderepressing the downstream caspase-3 protease.

Caspase activation also can involve cytochrome c, which in mammaliancells is often released from mitochondria into the cytosol duringapoptosis (Liu et al., Cell 86:147-157 (1996); Kharbanda et al., Proc.Natl. Acad. Sci., USA 94:6939-6942 (1997); Kluck et al., Science275:1132-1136 (1997); and Yang et al., Science 275:1129-1132 (1997)).Upon entering the cytosol, cytochrome c induces the ATP- ordATP-dependent formation of a complex of proteins that results inproteolytic activation of pro-caspase-3 and apoptotic destruction ofnuclei (Liu et al., supra, 1996). Among the members of this complex arethe CED-4 homolog Apaf-1, and caspase-9 (Apaf-3; Liu et al., supra,1996; Li et al., Cell 91:479-489 (1997); Zou et al., Cell 90:405-413(1997)). XIAP, c-IAP-1 and c-IAP-2 suppress apoptosis induced by stimuliknown to cause release of cytochrome c from mitochondria and can inhibitcaspase activation induced by cytochrome c in vitro. Thus, the agentsand methods of the invention can be used to allow apoptosis to occur inresponse to release of cytochrome c from mitochondria by suppressinginhibition of a caspase by XIAP, c-IAP-1 or c-IAP-2.

The invention further provides a method of reducing the severity of apathologic condition in an individual, by administering to an individualhaving a pathologic condition characterized by a pathologically reducedlevel of apoptosis, an effective amount of an agent to derepress anIAP-inhibited caspase. Examples of conditions characterized bypathologically reduced levels of apoptosis that can be treated in amethod of the invention include, but are not limited to, restenosis;autoimmune disease such as lupus or Rheumatoid Arthritis; allograftrejection, proliferative lesions of the skin such as Eczema; or benignprostate hypertrophy The agent can have a core motif selected from acore peptide of the invention, such as Core peptides 4 through 39 and 42through 55, or a core structure of the invention such as TPI 759, TPI882, TPI 914, TPI 927 or a compound comprising a core structure selectedfrom TPI 1391, TPI 1349, TPI 1396, TPI 1509, TPI 1540, TPI 1400, TPI 792and TPI 1332.

An effective amount of an agent that derepresses an IAP-inhibitedcaspase when used to treat a pathological condition is an amountrequired to allow an increase in apoptosis when administered to anindividual. The dosage of an agent of the invention required to betherapeutically effective will depend, for example, on the pathologicalcondition to be treated, the route and form of administration, theweight and condition of the individual, and previous or concurrenttherapies. The appropriate amount considered to be an effective dose fora particular application of the method can be determined by thoseskilled in the art, using the guidance provided herein. For example, theamount can be extrapolated from in vitro or in vivo assays as describedpreviously. One skilled in the art will recognize that the condition ofthe patient can be monitored throughout the course of therapy and thatthe amount of the agent that is administered can be adjustedaccordingly.

For treating or reducing the severity of a pathological condition, aneffective amount is an efficacious amount of the agent capable ofincreasing apoptosis that is pathologically reduced. An effective amountcan be, for example, between about 10 μg/kg to 500 mg/kg body weight,for example, between about 0.1 mg/kg to 100 mg/kg, or preferably betweenabout 1 mg/kg to 50 mg/kg, depending on the treatment regimen. Forexample, if an agent or formulation containing the agent is administeredfrom one to several times a day, then a lower dose would be needed thanif a formulation were administered weekly, or monthly or lessfrequently. Similarly, formulations that allow for timed-release of theagent, such as those described above, would provide for the continuousrelease of a smaller amount of derepressor of apoptosis than would beadministered as a single bolus dose. For example, an agent of theinvention can be administered at between about 1-5 mg/kg/week.

Formulations of a derepressor of an IAP-inhibited caspase, variants andcombinations thereof can also be delivered in an alternatingadministrations so as to combine their apoptosis increasing effects overtime. For example, an agent having a core peptide or structure of theinvention can be administered in a single bolus dose followed bymultiple administrations of one or more such agents species or variantalone, or in combination with a different formulation of such an agentor formulation of a different agent. Whether simultaneous or alternatingdelivery of the agent formulation, variant or combination thereof, themode of administration can be any of those types of administrationsdescribed previously and will depend on the particular therapeutic needand efficacy of the derepressor of an IAP-inhibited caspase selected forthe purpose. Determining which agent, formulation, species and variantsto combine in a temporally administered regime, will depend on thepathological condition to be treated and the specific physicalcharacteristics of the individual affected with the disease. Thoseskilled in the art will know or can determine a specific regime ofadministration which is effective for a particular application using theteachings and guidance provided herein together with diagnostic andclinical criteria known within the field of art of the particularpathological condition.

The methods of treating a pathological condition characterized bypathologically reduced apoptosis additionally can be practiced inconjunction with other therapies. For example, for treating cancer, themethods of the invention can be practiced prior to, during, orsubsequent to conventional cancer treatments such as surgery,chemotherapy, including administration of cytokines and growth factors,radiation or other methods known in the art.

Such treatments can act in a synergistic manner, with the reduction intumor mass caused by the conventional therapy increasing theeffectiveness of a compound of the invention, and vice versa.Non-limiting examples of anti-cancer drugs that are suitable forco-administration with a compound of the invention are well known tothose skilled in the art of cancer therapy and includeaminoglutethimide, amsacrine (m-AMSA), azacitidine, asparaginase,bleomycin, busulfan, carboplatin, carmustine (BCNU), chlorambucil,cisplatin (cis-DDP), cyclophosphamide, cytarabine HCl, dacarbazine,dactinomycin, daunorubicin HCl, doxorubicin HCl, erythropoietin,estramustine phosphate sodium, etoposide (V16-213), floxuridine,fluorouracil (5-FU), flutamide, hexamethylmelamine (HMM), hydroxyurea(hydroxycarbamide), ifosfamide, interferon alpha, interleukin 2,leuprolide acetate (LHRH-releasing factor analogue), lomustine (CCNU),mechlorethamine HCl (nitrogen mustard), melphalan, mercaptopurine,mesna, methotrexate (MTX), mitoguazone (methyl-GAG, methyl glyoxalbis-guanylhydrazone, MGBG), mitomycin, mitotane (o. p′-DDD),mitoxantrone HCl, octreotide, pentostatin, plicamycin, procarbazine HCl,semustine (methyl-CCNU), streptozocin, tamoxifen citrate, teniposide(VM-26), thioguanine, thiotepa, vinblastine sulfate, vincristinesulfate, vindesine sulfate, Herceptin, and MabThera. As set forth aboveand demonstrated by the results of Example X, TPI 792-33 or TPI 792-35can be administered in conjunction with VP-16 to treat cancer.Similarly, as demonstrated by the results of Example XIV, TPI 1396-34also can be administered in conjunction with an anti-cancer drug totreat cancer. Those skilled in the art will appreciate that similareffects are expected for any active polyphenylurea compound of theinvention.

Similarly, for treating pathological conditions which include infectiousdisease, the methods of the invention can be practiced prior to, during,or subsequent to conventional treatments, such as antibioticadministration, against infectious agents or other methods known in theart. Treatment of pathological conditions of autoimmune disorders alsocan be accomplished by combining the methods of the invention forderepressing an IAP-inhibited caspase with conventional treatments forthe particular autoimmune diseases. Conventional treatments include, forexample, chemotherapy, steroid therapy, insulin and other growth factorand cytokine therapy, passive immunity, inhibitors of T cell receptorbinding and T cell receptor vaccination. The methods of the inventioncan be administered in conjunction with these or other methods known inthe art and at various times prior, during or subsequent to initiationof conventional treatments. For a description of treatments forpathological conditions characterized by aberrant cell growth see, forexample, The Merck Manual, Sixteenth Ed, (Berkow, R., Editor) Rahway,N.J., 1992. Furthermore, anti-cancer drugs including, for example, anyof those set forth above with regard to combination compositions, can beadministered prior to, during, or subsequent to administration of aderepressor of an IAP-inhibited caspase in a method of treatment.

As described above, administration of a formulation of an agent thatderepresses an IAP-inhibited caspase can be, for example, simultaneouswith or delivered in alternative administrations with the conventionaltherapy, including multiple administrations. Simultaneous administrationcan be, for example, together in the same formulation or in differentformulations delivered at about the same time or immediately insequence. Alternating administrations can be, for example, delivering anagent of the invention and a conventional therapeutic treatment intemporally separate administrations. As described previously, thetemporally separate administrations of an agent of the invention andconventional therapy can similarly use different modes of delivery androutes.

A condition characterized by a pathologically reduced level of apoptosisthat can be treated using the agents and methods of the inventioninclude, for example, cancer, hyperplasia, autoimmune disease andrestenosis. A growing number of human diseases have been classified asautoimmune and include, for example, rheumatoid arthritis, myastheniagravis, multiple sclerosis, psoriasis, systemic lupus erythematosus,autoimmune thyroiditis, Graves disease, inflammatory bowel disease,autoimmune uveoretinitis, polymyositis and diabetes. Animal models formany conditions characterized by a pathologically reduced level ofapoptosis have been developed and can be employed for predictiveassessment of therapeutic treatments employing an agent that derepressesan IAP-inhibited caspase. Moreover, pharmaceutical compositions of aderepressor of IAP-inhibited caspase can be reliably extrapolated forthe treatment of these conditions from such animal models.

Those skilled in the art will know how to determine efficacy or amountsof an agent of the invention to administer based on the results ofroutine tests in a relevant animal model. The amount of an agent to beadministered can be determined in a clinical setting as well based onthe response in a treated individual. Modulation of efficacy, willdepend on the pathological condition and the extent to which progressionof apoptosis is desired for treatment or reduction in the severity ofthe pathological condition. Modulation can be accomplished by adjustingthe particular agent used to derepress an IAP-inhibited caspase,formulation, or dosing strategy. Based on the guidance provided herein,those skilled in the art will be able to modulate efficacy in responseto well known indicators of the severity of the particular conditionbeing treated. For a description of indicators for the variouspathological conditions described herein or otherwise known to becharacterized by a pathologically reduced level of apoptosis see, forexample, The Merck Manual, Sixteenth Ed, (Berkow, R., Editor) Rahway,N.J., 1992.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Identification of Derepressors of an IAP-Inhibited Caspasefrom Hexapeptide Libraries

This example demonstrates an IAP derepression assay. This Examplefurther demonstrates a positional-scanning approach to identifyingagents that are capable of derepressing an IAP-inhibited caspase.

The DCR390 library consisting of 120 mixtures of hexapeptides wassynthesized using methods known in the art as described in R. Houghtenet al. PCT/US91/08694 and U.S. Pat. No. 5,556,762. Each mixture was madeup of a population of hexapeptides all of which had the same amino acidat a defined position and any combination of the 20 essential aminoacids at the remaining 5 positions. Each mixture is identified by theposition number where the defined amino acid occurs (numbered from 1 to6 going from the amino-terminus to carboxy-terminus of the hexapeptide)and the identity of the defined amino acid. Thus, as shown in the firstcolumn of Table I, the mixture having a tryptophan at position 1 and allcombinations of the 20 amino acids at positions 2 through 5 isidentified as “position 1, W.”

The DCR390 library was screened using the assay set forth below. Basedon the mixtures identified from the DCR390 library screen as beingcapable of derepressing an IAP-inhibited caspase, additional definedpositions were incorporated into the TPI 1239 and TPI 1328 sublibraries,and the sublibraries screened using the same assay.

Caspase activity was assayed by release of7-amino-4-trifluoromethyl-coumarin (AFC) from Ac-DEVD-AFC syntheticpeptide using a Molecular Devices Spectromax 340 (see Zhou et al., J.Biol. Chem. 272:7797-7800 (1997)). Candidate mixtures were screened forthe ability to derepress an IAP-inhibited caspase by measuring AFChydrolysis rates for mixtures containing purified recombinant caspase-3,Ac-DEVD-AFC, and GST-XIAP in the presence and absence of the candidateagent. The ratio of V_(max) for hydrolysis of Ac-DEVD-AFC in thepresence and absence of the candidate mixture was calculated and used toidentify those that contain an agent that derepresses an IAP-inhibitedcaspase. The ratio=(V_(max) when candidate mixture, caspase 3 and XIAPare present)/(V_(max) when caspase 3 and XIAP are present).

Screening of each mixture from the DCR390, TPI 1239 or TPI 1328 library,respectively, using the above described assay was carried out asfollows. Each mixture was aliquoted in a 25 microliter volume and induplicate to a well of a 96 well microtiter plate. Into the first set ofduplicate wells was added 25 microliters of caspase assay buffer (50 mMHEPES, pH 7.4, 100 mM NaCl, 10% sucrose, 10 mM DTT, 1 mM EDTA and 0.1%CHAPS) and into the second set of wells was added 25 microliters of astock solution of 40 nM XIAP in caspase assay buffer. Each microtiterplate also had the following controls: (1) a buffer blank well to whichwas added 25 microliters of caspase assay buffer and 25 microliters ofpeptide carrier solvent, (2) an XIAP control well to which was added 25microliters of peptide carrier solvent and 25 microliters of a stocksolution of 40 nM XIAP in caspase assay buffer, and (3) a SMAC controlwell to which was added 25 microliters of a stock solution of 40 nM XIAPin caspase assay buffer, and 2.5 microliters of 4 μM SMAC peptide(H-Ala-Val-Pro-Ile-Ala-Gln-Lys-NH₂, SEQ ID NO:5). Into each of thesample and control wells was added 25 microliters of 0.64 nM caspase-3solution followed by 25 microliters of 400 μM Ac-DEVD-AFC substrate.Fluorescence of liberated AFC was immediately detected from each wellfor 30 minutes at 30 second intervals. The V_(max) for hydrolysis ofAC-DEVD-AFC from each well was measured using the softmax softwarepackage.

Results of the screen for the DCR390 library are shown in Table I. Setsof mixtures having the same position fixed with different respectiveamino acids are arranged in 6 sections identified with the positionnumber. Within each of the six sections are 3 columns showing (1) theidentity of the fixed amino acid, (2) the apparent velocity of thereaction when candidate mixture, caspase 3 and XIAP are present, and (3)the apparent velocity of the reaction when candidate mixture, andcaspase 3 are present. Also shown in each section are results for theXIAP control reaction. The mixtures in each section are arranged indescending order according to apparent velocity in the second column.Those mixtures having significantly higher apparent velocities comparedto the XIAP control reaction are listed above the horizontal line andare thereby identified as containing an agent capable of derepressing anXIAP-inhibited caspase. TABLE I DCR 390 Amino Acid XIAP + MixturesMixtures Position 1 W 43 63 A 35 67 Y 35 63 F 34 61 C 30 62 L 26 63 I 2364 E 22 64 T 22 62 V 20 60 M 19 65 G 17 65 P 17 64 Q 17 66 XIAP 16 64 R16 65 XIAP 15 64 H 15 63 K 15 65 S 15 65 D 14 67 N 13 63 XIAPX1.5 24Position 2 W 57 58 F 44 58 L 38 61 C 36 64 I 32 61 V 30 59 Y 30 61 A 2062 D 20 67 P 20 60 R 20 58 XIAP 19 63 XIAP 19 63 G 19 58 H 19 58 M 19 60E 18 67 N 18 64 T 18 59 Q 17 59 S 17 59 K 16 61 XIAPX1.5 29 Position 3 F50 64 I 36 65 W 34 65 L 26 65 C 20 64 V 19 62 A 16 67 H 16 70 K 15 67 Y14 63 XIAP 13 64 XIAP 13 64 D 13 66 T 13 62 M 12 67 N 12 64 R 12 67 E 1163 G 11 63 P 11 67 S 11 68 Q 10 63 XIAPX1.5 20 Position 4 W 52 63 F 3761 L 23 61 Y 17 64 C 15 60 I 15 60 V 13 63 XIAP 12 61 M 12 64 N 12 63 A11 64 XIAP 10 61 D 10 63 E 10 59 G 10 60 H 10 59 P 10 66 Q 10 64 S 10 62K 9 60 R 9 62 T 9 62 XIAPX1.5 18 Position 5 W 44 56 Y 23 57 V 15 57 I 1457 L 14 52 A 13 57 C 12 54 F 12 53 XIAP 11 52 XIAP 11 52 H 11 57 K 11 55S 11 57 T 11 56 D 10 54 E 10 53 M 10 49 P 10 54 G 9 57 N 9 52 Q 9 54 R 952 XIAP1.5 17 Position 6 W 23 51 R 22 52 A 13 53 C 12 51 G 12 51 K 12 53Q 11 50 XIAP 10 51 XIAP 10 51 S 10 53 Y 10 51 D 9 50 E 9 49 L 9 53 M 951 N 9 50 T 9 53 V 9 51 F 8 50 H 8 51 I 8 53 P 8 49 XIAPX1.5 15

Based on the results of the DCR390 screen, the TPI 1239 library wassynthesized and screened using the above-described caspase assay. Inparticular mixtures were synthesized having positions 5 and 6 defined astryptophan, positions 3 and/or 4 defined variously, and the remainingpositions randomized with the 20 essential amino acids as set forth inTable II. As shown in Table II, in the absence of XIAP the mixtures hadan insignificant effect on caspase activity. Mixtures having ratios of1.9 or higher in the presence of XIAP were identified as containing anagent capable of derepressing an IAP-inhibited caspase. TABLE II TPI1239 V_(max)(mix + casp3) V_(max)(mix + casp3 + XIAP) MixtureV_(max)(casp3) V_(max)(casp3 + XIAP) caspase 3 1.0 ± 0.0 5.1 ± 2.3 Xiap+ caspase3 0.2 ± 0.1 1.0 ± 0.0 SMAC 0.8 ± 0.0 3.8 ± 1.7 XXFWWW SEQ IDNO: 11 0.9 ± 0.0 0.8 ± 0.1 XXLWWW SEQ ID NO: 12 0.9 ± 0.0 0.7 ± 0.1XXWLWW SEQ ID NO: 13 0.9 ± 0.0 0.7 ± 0.1 XXWWWW SEQ ID NO: 14 0.9 ± 0.00.8 ± 0.1 XXXTWW 0.8 ± 0.0 4.2 ± 1.6 XXXAWW 0.9 ± 0.0 3.7 ± 1.6 XXXSWW0.8 ± 0.0 3.4 ± 1.1 XXXQWW 0.8 ± 0.0 2.4 ± 0.4 XXXKWW 0.9 ± 0.0 2.3± 1.0 XXXVWW 0.9 ± 0.0 2.2 ± 0.5 XXXRWW 0.9 ± 0.0 2.1 ± 0.2 XXXHWW 0.9± 0.0 2.1 ± 0.6 XXXNWW 0.9 ± 0.0 1.9 ± 0.6 XXXPWW 0.9 ± 0.0 1.5 ± 0.2XXXYWW 0.9 ± 0.0 1.2 ± 0.3 XXXDWW 0.9 ± 0.0 1.1 ± 0.2 XXXIWW 0.9 ± 0.00.9 ± 0.1 XXXLWW 0.9 ± 0.0 0.9 ± 0.1 XXXCWW 0.9 ± 0.0 0.8 ± 0.2 XXXEWW0.9 ± 0.0 0.8 ± 0.2 XXXGWW 0.9 ± 0.0 0.7 ± 0.1 XXXMWW 0.9 ± 0.0 0.6± 0.2 XXXFWW 0.9 ± 0.0 0.6 ± 0.1 XXXWWW 0.8 ± 0.0 not determined XXXXWW1.0 ± 0.0 2.0 ± 0.3

The mixtures identified from the TPI 1239 library as containing an agentcapable of derepressing an IAP-inhibited caspase were further analyzedfor dose response. The dose response data is provided in Figure

11 which shows that the mixtures had no effect on caspase activity. Themost active mixtures were found to have alanine, lysine or threonine atposition 4, tryptophan at positions 5 and 6 and mixtures at positions 1through 3.

Based on the results of the DCR390 and TPI 1239 library screens, the TPI1328 library was synthesized and screened using the above-describedcaspase assay. For each mixture in the TPI 1328 library, 3 to 4positions were defined and the remaining positions werecombinatorialized with all 20 of the essential amino acids includingAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, Cys or Tyr. Position 4 was defined with Ala, Lys ora mixture of Ala, Lys and Thr (the mixture is referred to as “3X” or“A,K,T”).

Various TPI 1328 sublibraries that were screened and values obtained forthe ratio of V_(max) for hydrolysis of Ac-DEVD-AFC in the presence andabsence of each mixture are plotted in FIG. 1. In FIG. 1 and Table III,“X” represents a mixture of all 20 essential amino acids and “0”represents the location of the defined position, the identity of theamino acid at the defined position being plotted on the x axis.Candidates having a ratio over 1.7 were identified as being derepressorsof XIAP-inhibited caspase-3. A list of derepressors of XIAP-inhibitedcaspase-3 identified from the TPI 1328 library is provided in Table III.TABLE III Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 SEQ ID NO: X X Ala Ala TrpTrp 7 X X Gly Ala Trp Trp 8 X X Arg Ala Trp Trp 9 X X X Ala Trp Trp X XCys Lys Trp Trp 10 X X Leu Lys Trp Trp 15 X X Gly 3X Trp Trp X X Arg 3XTrp Trp X X Thr 3X Trp Trp X X Val 3X Trp Trp X Thr X 3X Trp Trp X Tyr X3X Trp Trp Ala X X 3X Trp Trp Cys X X 3X Trp Trp Phe X X 3X Trp Trp LysX X 3X Trp Trp

EXAMPLE II Identification of Derepressors of an IAP-Inhibited Caspasefrom the TPI 1332 and TPI 1352 Individual Tetrapeptide Libraries

This Example demonstrates identification of agents from the TPI 1332 andTPI 1352 tetrapeptide libraries that are capable of derepressing anXIAP-inhibited caspase-3.

The TPI 1332 and TPI 1352 tetrapeptide libraries were synthesizedidentically with the exception that the formyl protecting groups ontryptophan were removed by different procedures. The deprotection stepused for the TPI 1332 library was less complete leaving the possibilitythat some of the tryptophan residues present on candidate compounds usedin the screen retained formyl protecting groups. The deprotectingchemistry used for the TPI 1352 library was substantially complete,however resulted in the formation of polymeric structures for a subsetof the species in the library.

Candidates from the TPI 1332 and TPI 1352 libraries were screened usingthe derepression assay described in Example I. The ratios of V_(max) forhydrolysis of Ac-DEVD-AFC in the presence and absence of each species ofthe TPI 1332 and TPI 1352 libraries were determined and those havingvalues greater than 2.4 were identified as derepressors of anIAP-inhibited caspase.

A list of derepressors of XIAP-inhibited caspase-3 identified from theTPI 1332 and TPI 1352 tetrapeptide libraries is provided in Table IV.Agents identified in both libraries are indicted as “1332/1352”. TABLEIV Agent Position 1 Position 2 Position 3 Position 4 1332/1352-1 L-AlaL-Trp L-Trp L-ThiAla 1332/1352-2 L-Ala L-Trp L-Trp L-pClPhe 1332/1352-47L-Ala D-Trp L-Trp L-ThiAla 1332-13 L-Ala D-Nal L-Trp L-Nal 1332-24 D-TrpD-Trp L-Trp D-Nal 1332-41 L-Cha D-Nal L-Trp L-ThiAla 1352-5 L-Ala L-TrpL-Trp L-3I-Tyr 1352-6 L-Ala D-Trp L-Trp L-ThiAla 1352-32 L-Cha L-TrpL-Trp L-pClPhe 1352-46 L-Ala D-Trp L-Trp D-Trp 1352-48 L-Ala D-Trp D-PheD-Trp 1352-64 L-Nal D-Trp D-Phe D-Trp 1352-66 L-Nal D-Cha L-Trp D-Trp1352-72 L-Nal D-ThiAla D-Phe D-Trp

Structures of TPI 1332 library compounds are shown in FIG. 36A;structure of related compounds of the TPI 1495 series are shown in FIG.37.

EXAMPLE III Identification of Individual Peptide Derepressors of anIAP-Inhibited Caspase from the TPI 792 Library

This Example demonstrates identification of agents from the TPI 792library that are capable of derepressing an XIAP-inhibited caspase-3.

The TPI 792 library is based on a tetrapeptide backbone. The species ofthe TPI 792 library were screened in the derepression assay described inExample I. A list of derepressors of XIAP-inhibited caspase-3 identifiedfrom the TPI 792 library is provided in Table V. Structures for the TPI792 core peptides that were tested are shown in FIG. 20. TABLE V LCAgent Pos 1 Pos 2 Pos 3 Pos 4 μg/ml 792-3 D-Nal Lys-εFmoc L-pClPheLys-εFmoc 2 792-9 D-Nal D-pClPhe L-pClPhe Lys-εFmoc 10 792-15 D-NalL-Nal L-pClPhe D-Lys-εFmoc 2 792-17 D-Nal L-Nal D-Lys(Fm) Lys-εFmoc 2792-19 L-ThiAla Lys-εFmoc D-Nal Lys-εFmoc 2 792-22 L-ThiAla Lys-εFmocL-pClPhe D-pFPhe 2 792-27 L-ThiAla D-pClPhe L-pClPhe Lys-εFmoc 2 792-33L-ThiAla L-Nal L-pClPhe Lys-εFmoc 0.4 792-35 L-ThiAla L-Nal D-Lys(Fm)D-Lys-εFmoc 2

The dose response of the agents identified from the TPI 792 library weredetermined by repeating the derepression assay with variableconcentrations of the agent. Four concentrations were chosen: 0.4, 2, 10and 50 micrograms per milliliter. From this data the lowestconcentration with a ratio of 2 or higher in the derepression assay (LC)was determined and shown in Table V. The lowest LC value determined fromthe TPI 792 library was 0.4 micrograms per milliliter for TPI792-33.

EXAMPLE IV Identification of Derepressors of an IAP-Inhibited Caspasefrom the TPI 1313 Library

This Example demonstrates identification of agents from the TPI 1313library that are capable of derepressing an XIAP-inhibited caspase-3.

The TPI 1313 library is based on a tetrapeptide backbone. The species ofthe TPI 1313 library, listed in FIG. 2 and shown in FIG. 3, werescreened in the derepression assay described in Example I. A list ofderepressors of XIAP-inhibited caspase-3 identified from the TPI 1313library is provided in Table VI. TABLE VI Agent Pos 1 Pos 2 Pos 3 Pos 41313-4 L-ThiAla D-pCL-Phe D-Nal D-pCL-Phe 1313-5 L-ThiAla D-pCL-PheD-Nal D-pNO₂Phe 1313-7 L-ThiAla D-OEt-Tyr D-OEt-Tyr D-pCL-Phe 1313-40Phe D-pCL-Phe D-Nal D-pCL-Phe

The dose response of the agents identified from the TPI 1313 librarywere determined by repeating the derepression assay with variableconcentrations of the agent. Four concentrations were chosen: 0.4, 2, 10and 50 micrograms per milliliter. From this data the apparent IC₅₀ wasdetermined. The lowest IC₅₀ value determined from the TPI 1313 libraryrange from 3.9 to 6.3 micrograms per milliliter for TPI 1313-7.

EXAMPLE V Identification of Derepressors of an IAP-Inhibited Caspasefrom the TPI 1325 Library

This Example demonstrates identification of agents from the TPI 1325library that are capable of derepressing an XIAP-inhibited caspase-3.

The TPI 1325 library was screened in the derepression assay described inExample I. For each species aliquoted in the assay 1 position was fixed(i.e. having a single known amino acid R group) and three positions werecombinatorialized. Thus, each “mixture” identified from the TPI 1325library represents a mixture of compounds where X₁ and X₂ are selectedfrom Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp and Tyr and X₃ includes any one of Ala, Lys andThr.

A list of derepressors of XIAP-inhibited caspase-3 identified from theTPI 1325 library is provided in Table VII. TABLE VII Mixture Pos 1 Pos 2Pos 3 Pos 4 1325-10 L-Ala L-Met X₁ X₂ 1325-15 L-Ala L-Ser X₁ X₂ 1325-16L-Ala L-Thr X₁ X₂ 1325-18 L-Ala L-Trp X₁ X₂ 1325-44 L-Ala L-ThiAla X₁ X₂1325-61 L-Ala X₁ X₂ X₃ 1325-64 X₁ X₂ L-Trp D-Trp

The dose response of the agents identified from the TPI 1325 librarywere determined by repeating the derepression assay with variableconcentrations of the agent. Four concentrations were chosen: 0.4, 2, 10and 50 micrograms per milliliter. From this data the apparent IC₅₀ wasdetermined. The lowest IC₅₀ value determined from the TPI 1325 librarywas 12 micrograms per milliliter for TPI 1325-15.

EXAMPLE VI Identification of Derepressors of an IAP-Inhibited Caspasefrom the TPI 914, TPI 927, TPI 759 and TPI 882 Libraries

This Example demonstrates identification of agents, from non-peptidebased libraries, that are capable of derepressing an XIAP-inhibitedcaspase-3.

The TPI 914, TPI 927, TPI 759 and TPI 882 libraries were screened usingpositional scanning (as described in U.S. Pat. No. 5,556,762) incombination with the derepression assay described in Example I.

Analysis was started with combinatorial libraries in which at least oneposition was defined. Hits were identified as those mixtures producing amixture/XIAP ratio that was greater than or equal to 2. Followinganalysis of the first library, libraries of increasing definition werescreened until a discrete library was prepared in which all positionswere defined. Hits from this defined library were then checked for adose response which yielded the IC50 values listed below.

The TPI 914 N-acyltriamine library included 50 amino acid R groups atposition R1, 50 amino acid R groups at position R2 and 50 acidderivatives at position R3 for a total diversity of 125,000 species.Mixtures having a defined functionality at one of the R positions andidentified by positional scanning of the TPI 914 library as having apeptide/XIAP ratio greater than or equal to about 1.8 when present at 25micrograms per milliliter in the derepression assay were identified andare shown in FIG. 4. Control agents having a peptide/XIAP ratio greaterthan or equal to about 1.8 when present at 6.25 micrograms permilliliter or 12.5 micrograms per milliliter in the derepression assaywere identified and are shown in FIG. 5. Additional compounds designedbased on this screening are shown in FIG. 21A as TPI 1349-1 through TPI1349-34. The activity of these compounds is shown in FIGS. 21B-D.

The TPI 927 polyphenylurea library included 48 amino acid R groups atposition R1, 48 amino acid R groups at position R2 and 39 acidderivatives at position R3 for a total diversity of 89,856 species.Mixtures having a defined functionality at one of the R positions andidentified by positional scanning of the TPI 927 library as having apeptide/XIAP ratio greater than or equal to about 1.8 when present at 4micrograms per milliliter in the derepression assay were identified andare shown in FIG. 6. Control agents having a peptide/XIAP ratio greaterthan or equal to about 1.8 when present at 25 micrograms per milliliterin the derepression assay were identified and are shown in FIG. 9.

In particular, as shown in FIG. 25 a, aliquots of the mixture-basedcombinatorial library of poly-phenylureas based on TPI 927 describedabove were added to microtiter plates containing XIAP and caspase-3(black bars) or, as a control, caspase-3 alone (gray bars). Caspase-3activity was measured by monitoring cleavage of the fluorogenicsubstrate Ac-DEVD-AFC as described herein. Briefly, recombinant proteinswere produced in bacteria and purified as described, for example, inDeveraux et al., Nature 388:300-304(1997). GST-XIAP (46 nM) was added toactive caspase-3-His6 (0.36 nM) in 100 μl of 50 mM HEPES pH 7.4, 10%sucrose, 1 mM EDTA, 0.1% CHAPS, 100 mM NaCl, and 10 mM DTT to achieveapproximately 75% inhibition of protease activity. Activity of caspase-3was measured by monitoring cleavage of the fluorogenic tetrapeptidesubstrate acetyl-DEVD-AFC (BIOMOL, Plymouth, Pa.) at 100 μM. Generationof fluorogenic AFC (7-amino-4-trifluoromethyl coumarin) product wasmeasured with a spectrofluorometric plate reader in kinetic mode for 30minutes at 37° C. using excitation and emission wavelengths of 405 nmand 510 nm, respectively. Chemical compounds were screened at 6.25, 12.5and 25.0 μg/ml to identify compounds that increase caspase-3 inducedcleavage of Ac-DEVD-AFC. Control reactions lacked XIAP, and all assayswere conducted in the linear range of substrate hydrolysis to avoidsubstrate depletion artifacts.

A representative screen of the positional scanning combinatorial library(final concentration 25 mg/ml) is shown in FIG. 25 a. In FIG. 25 a, hitswere defined as compounds that increased caspase-3 activity greater thanor equal to 2 fold in XIAP-inhibited reactions without affectingcaspase-3 alone. Caspase activity is presented as the fold increase inenzyme velocity after the addition of the compound. The positivecompound mixtures were deconvoluted by standard methods yielding 36individual compounds which were screened in the same caspasederepression assay as described below.

The 36 individual compounds (TPI 1396-1 through TPI 1396-36) weresynthesized based on deconvolution of the polyphenylurea library. Theindividual compounds result from the combination of the definedfunctionalities of the most active mixtures of the positional scanningcombinatorial library. The number of functionalities used were 3, 4, and3 at R1, R2 and R3, respectively. As shown in FIG. 25 b, each of the 36individual compounds was tested at 25 mg/ml using the caspasederepression assay for their ability to increase caspase-3 activity inthe presence (black bars) or absence (gray bars) of XIAP, using a 2-foldelevation in the enzyme velocity as the cut-off for positivity. Othercut-offs for positivity can include, for example, 1.5 fold and higher,or 1.8 fold and higher.

The TPI 759 N-benzyl-1,4,5-trisusbstituted-2,3-diketopiperazine libraryincluded 29 amino acid R groups at position R1, 27 amino acid R groupsat position R2 and 40 acid derivatives at position R3 for a totaldiversity of 31,320 species. Mixtures having a defined functionality atone of the R positions and identified by positional scanning of the TPI759 library as having a peptide/XIAP ratio greater than or equal toabout 2.0 (or in the case of the sublibrary where R3 was fixed, a ratioof 1.9 or higher) when present at 25 micrograms per milliliter in thederepression assay were identified and are shown in FIG. 8. Additionalcompounds designed based on these functionalities are shown in FIG. 23Aas TPI 1391-1 through TPI 1391-36. The activity of these compounds isshown in FIGS. 23B-F.

The TPI 882 C-6-acylamino bicyclic guanidine library included 43 aminoacid R groups at position R1, 41 acid derivatives at R2 and 41 acidderivatives at R3 for a total diversity of 72,283 species. Mixtureshaving a defined functionality at one of the R positions and identifiedby positional scanning of the TPI 882 library as having a peptide/XIAPratio greater than or equal to about 1.9 when present at 5 microgramsper milliliter in the derepression assay were identified and are shownin FIG. 7. Control agents having a peptide/XIAP ratio greater than orequal to about 2.0 when present at 8 micrograms per milliliter in thederepression assay were identified and are shown in FIG. 10. Additionalcompounds designed based on these functionalities are shown in FIG. 24Aas TPI 1400-1 through TPI 1400-58. The activity of these compounds isshown in FIGS. 24B-H.

EXAMPLE VII SMAC Competition Assay

This Example describes an assay useful for determining the bindingaffinity of a derepressor of an IAP-inhibited caspase for an IAP, orfunctional fragment thereof.

A polarization based binding assay was used to detect binding betweenrhodamine labeled SMAC (rhodamine-SMAC) and the XIAP fragments BIR2 orBIR3RING The assay is based on the decrease in mobility that occurs forrhodamine-SMAC when associated with XIAP or functional fragments thereofwhich is detected as a reduction in polarization for boundrhodamine-SMAC compared to free (unbound) rhodamine-SMAC.

Binding affinity of rhodamine-SMAC for a glutathione-S-transferase-BIR2fusion protein (GST-BIR2) or BIR3RING was determined as follows. Assayswere run in 50 mM Tris @ pH 7.2/100 mM NaCl/0.1% BSA. Rhodamine labeledSMAC was present at 400 nM. GST-BIR2 ranged from 0.05 to 20 μM whileGST-BIR3RING ranged from 0.02 to 6 μM. Plates (proxi from Packard) wereread in fluorescence polarization mode after 1 hr at 28° C. in a Victorfrom Perkin-Elmer with excitation at 531 nm and emission at 595 nm. Datawas plotted as a function of millipolars vs. protein concentration.Rhodamine-SMAC had a K_(d) of 20 μM for GST-BIR2 and 280 nM forGST-BIR3RING.

Unlabelled SMAC was titrated against a solution containing 400 nMrhodamine-SMAC and 10 μM GST-BIR2 or 1 μM GST-BIR3RING. Plates (proxifrom Packard) were read in fluorescence polarization mode after 1 hr at28° C. in a Victor from Perkin-Elmer with excitation at 531 nm andemission at 595 nm. Unlabeled SMAC was titrated in the range of 0 to 50μM Data was plotted as a function of millipolars vs SMAC concentrationand IC₅₀ values determined. The IC₅₀ value of the SMAC titration was 21μM for GST-BIR3RING. Competition with unlabeled SMAC was also seen forGST-BIR2 but was not sufficient to allow calculation of an IC₅₀.

Candidate agents from a library are added to a solution containing 400nM rhodamine-SMAC and 10 μM GST-BIR2. Fluorescence polarization isdetermined for each sample and those candidates that show a decrease inpolarization compared to a control reaction containing 400 nMrhodamine-SMAC and 10 μM GST-BIR2 are identified as derepressors of anIAP-inhibited caspase. As a control, fluorescence polarization is alsodetermined for the library sample in the absence of GST-BIR2.

An agent identified as a derepressor of an IAP-inhibited caspase istitrated against a solution of rhodamine-SMAC and GST-BIR2. Polarizationis determined at each concentration of the agent as described above.Data is plotted as a function of millipolars vs. agent concentration andbinding constants determined also as described above.

EXAMPLE VIII Screening of Individual Compounds from Various Libraries

This example describes screening of individual agents derived from TPI914, TPI 927, TPI 759 and TPI 882 libraries and identification ofindividual agents that Derepress an IAP-Inhibited Caspase.

Individual agents were synthesized based on the active agents identifiedin Example VI. Selected agents based on the TPI 914 derepressors shownin FIG. 5 were synthesized and are identified as agents TPI 1349-1through TPI 1349-34 in FIG. 21. Selected agents based on the TPI 927derepressors shown in FIG. 9 were synthesized and are identified asagents TPI 1396-1 through TPI 1396-65 in FIG. 22. Selected agents basedon the TPI 759 derepressors shown in FIG. 8 were synthesized and areidentified as agents TPI 1391-1 through TPI 1391-36 in FIG. 23. Selectedagents based on the TPI 882 derepressors shown in FIG. 10 weresynthesized and are identified as agents TPI 1400-1 through TPI 1400-58in FIG. 24.

The caspase derepression assay was used to evaluate the agents shown inFIGS. 21-24. Each compound was tested using the caspase derepressionassay for its ability to increase caspase-3 activity. The structures forTPI 1349-1 through TPI 1349-34 along with respective molecular weightsand masses are shown in FIG. 21A. The activity of TPI 1349-1 through TPI1349-34 in a caspase derepression assay using full length XIAP is shownin FIG. 21B. The activity of TPI 1349-1, -3, -8, -13, -23, and -28 usingboth full length XIAP and XIAP BIR2 domain is shown in FIG. 21C. Theactivity of TPI 1349-1, -3, -8, -13, -23, and -28 using cIAP-1 BIR2domain is shown in FIG. 21D. These data indicate that the TPI 1349compounds are active in derepressing caspase inhibited by either XIAP orthe BIR2 domain of XIAP, but do not overcome cIAP1-mediated suppressionof caspase-3. It is important to note that the lack of activity observedfor various compounds can be the result of the compounds being presentat a two fold excess over cIAP1.

The structures of TPI 1396-1 through TPI 1396-65 along with respectivemolecular weights and masses are shown in FIG. 22A. The activity of TPI1396-1 through TPI 1396-36 in a caspase derepression assay using fulllength XIAP is shown in FIG. 22B. The activity of TPI 1396-37 throughTPI 1396-65 in the derepression assay using full length XIAP is shown inFIG. 22C. A table indicating the activities of TPI 1396-11, -12, -22,-28, and -34 in the derepression assay using full length XIAP and theXIAP BIR2 domain is shown in FIG. 22D. The activity of TPI 1396-11, -12,-22, -28, and -34 at 50 μg/ml using XIAP BIR2 domain is shown in FIG.22E. Additional representative data for the activity of TPI 1396-11,-12, -22, -28, and -34 at 100 μg/ml using full length XIAP and Caspase 3or 7 is shown in FIG. 22F. The activity of TPI 1396-11, -12, -22, -28,and -34 at 100 μg/ml using cIAP-1 BIR2 domain is shown in FIG. 22G Thesedata indicate that TPI 1396 compounds are active in derepressing caspaseinhibited by either XIAP or the BIR2 domain of XIAP, but do not overcomecIAP1-mediated suppression of caspase-3. It is important to note thatthe lack of activity observed for various compounds can be the result ofthe compounds being present at a two fold excess over cIAP1.

The structures of TPI 1391-1 through TPI 1391-36 along with respectivemolecular weights and masses are shown in FIG. 23A. The activity of TPI1391-1 through TPI 1391-36 at 100 μg/ml in a caspase derepression assayusing full length XIAP is shown in FIG. 23B. The activity of TPI 1391-1through TPI 1391-36 at 25 μg/ml in the derepression assay using fulllength XIAP is shown in FIG. 23C. A table indicating the activities ofTPI 1391-1, -4, -5, 7, -17, -21, -25, -28, -34 and -35 in thederepression assay using full length XIAP is shown in FIG. 23D. Acomparison of the activities of TPI 1391-1, -4, -5, 7, -17, -21, -25,-28, -34 and -35 in the derepression assay using full length XIAP orXIAP BIR2 domain is shown in FIG. 23E. The activity of TPI 1391-1, -4,-5, 7, -17, -21, -25, -28, -34 and -35 using cIAP-1 BIR2 domain is shownin FIG. 23F. These data indicate that TPI 1391 compounds are active inderepressing caspase inhibited by either XIAP or the BIR2 domain ofXIAP, but do not overcome cIAP1-mediated suppression of caspase-3. It isimportant to note that the lack of activity observed for variouscompounds can be the result of the compounds being present at a two foldexcess over cIAP1.

The structures of TPI 1400-1 through TPI 1400-58 along with respectivemolecular weights and masses are shown in FIG. 24A. The activity of TPI1400-1 through TPI 1400-28 at 25 μg/ml in a caspase derepression assayusing full length XIAP is shown in FIG. 24B. The activity of TPI 1400-1through TPI 1400-28 at 10 μg/ml in the derepression assay using fulllength XIAP is shown in FIG. 24C. The activity of TPI 1400-29 throughTPI 1400-58 at 25 μg/ml in the derepression assay using full length XIAPis shown in FIG. 24D. The activity of TPI 1400-29 through TPI 1400-58 at10 μg/ml in the derepression assay using full length XIAP is shown inFIG. 24E. A table indicating the activities of TPI 1400-6, -7, 13, -14,-33, -37, -43, -44 in the derepression assay using full length XIAP isshown in FIG. 24F. A comparison of the activities of TPI 1400-6, -7, 13,-14, -33, -37, -43, -44 in the derepression assay using full length XIAPor XIAP BIR2 domain is shown in FIG. 24G The activity of TPI 1400-6, -7,13, -14, -33, -37, -43, -44 using cIAP BIR2 domain is shown in FIG. 24H.These data indicate that TPI 1400 compounds are active in derepressingcaspase inhibited by either XIAP or the BIR2 domain of XIAP, but do notovercome cIAP1-mediated suppression of caspase-3. It is important tonote that the lack of activity observed for various compounds can be theresult of the compounds being present at a two fold excess over cIAP1.

EXAMPLE IX Peptidyl and Non-Peptidyl Compounds Restore Caspase Activityof IAP-Inhibited Caspase In Vitro

This example demonstrates an assay for determining potency of peptidyland non-peptidyl derepressors of IAP-inhibited caspases in vitro. Thisexample also identifies peptidyl and non-peptidyl compounds havingpotency at restoring caspase activity of IAP-inhibited caspase in vitro.

The caspase derepression assay was used to evaluate peptidyl IAPantagonists identified from screens of the TPI 792 library andnon-peptidyl IAP antagonists identified from screens of the TPI 1391 andTPI 1396 libraries. Each compound was titrated against a solution ofrhodamine labeled SMAC tetrapeptide, AVPI (SEQ ID NO:4), and full lengthXIAP under the conditions described in Example VII. Polarization wasdetermined at each concentration of the IAP antagonist, data was plottedas a function of millipolars vs. compound concentration, and the EC50binding constants were determined from the plots. As a control unlabeledSMAC tetrapeptide, AVPI (SEQ ID NO:4) was also assayed.

For several compounds of the TPI 1396 library (TPI 1396-11, -12, -22,-28, and -34), the caspase derepression assay was carried out in thepresence of caspase-3 or caspase-7. These studies, representativeresults of which are shown in FIG. 22F, polyphenylureas reversedXIAP-mediated suppression of caspases 3 and 7. For these experiments,GST-XIAP was added to active caspase-3 (0.69 nM) or caspase 7 (3.2 nM)and 75 μM compounds with 100 μM DEVD-AFC in a 100 μl of buffer.Generation of AFC was measured in a spectrofluorimeter with 405 nmexcitation and 510 nm emission at 37° C. for 30 minutes. The data shownin FIG. 22F represent caspase activity, compared to XIAP-inhibitedreactions (=1.0) and are mean±standard deviation of threedeterminations. As a control unlabeled SMAC heptapeptide, AVPIAQK wasalso assayed.

Table VIII summarizes the results of the SMAC competition assay for IAPantagonists identified from the TPI 792, TPI 1391 and TPI 1396libraries. The EC50 was determined, by calculating the amount ofcompound necessary to restore caspase-3 activity to 50% of maximumvelocity (Vmax). Two of the most potent tetramer peptides were TPI792-33 and TPI 792-35 which displayed enzyme derepression activities invitro that were 5.2 to 2.5 fold better than SMAC peptide, respectively.The most potent diketopiperazine based compounds included TPI 1391-21,TPI 1391-28 and TPI 1391-34 which exhibited potencies 3.3 to 5.0 foldmore active than SMAC peptide. The most potent phenyl-urea compoundsincluded TPI 1396-22, TPI 1396-34 and TPI 1396-28 which exhibitedpotencies that were 1.6 to 2.8 fold more active than SMAC peptide.

The caspase derepression assay was used to evaluate non-peptidyl IAPantagonists identified from screens of the TPI 1396 library in thepresence of the cIAP1 BIR2 domain. Each compound was present at a 100 μMwith caspase-3 at 8.5 nM, cIAP BIR2 at 37 μM and 100 μM Ac-DEVD-AFC.Assays were initiated upon addition of DEVD substrate and release offluorogenic product was followed in the kinetic mode for 30 minutes at37° C. Assays were performed in a Molecular Devices FMAXspectrofluorimeter. Ratios are relative to assay with cIAP BIR2 in theabsence of compound. FIGS. 22B and 22C show relative caspase activity inthe presence of various TPI 1396 library compounds. As is shown in FIG.22G, polyphenylurea compounds inhibit XIAP but do not inhibit cIAP1, asassayed using the cIAP1 BIR2 domain. Compounds TPI 1396-11, TPI 1396-12,TPI 1396-22 and TPI 1396-34 represent active XIAP inhibitors, while TPI1396-28 is an inactive analog. TABLE VIII Relative Natural peptides EC50(μM) Potency SMAC AVPI tetrapeptide (SEQ ID NO: 4) 125 1.0 Un-naturalpeptides TPI 792-33 24 5.2 TPI 792-35 51 2.5 Diketopiperazines TPI1391-21 33.6 3.7 TPI 1391-28 25.1 5 TPI 1391-34 39.4 3.3 Diphenyl andTriphenyl Ureas TPI 1396-22 45.3 2.8 TPI 1396-34 77.1 1.6 TPI1396-28 >134 N/A

These results demonstrate that peptidyl compounds TPI 792-33 and TPI792-35; diketopiperazine based compounds TPI 1391-21, TPI 1391-28 andTPI 1391-34; and phenyl-urea compounds TPI 1396-22 and TPI 1396-34derepressed XIAP inhibited caspase in vitro and did so with more potencythan the SMAC AVPI tetrapeptide (SEQ ID NO:4).

EXAMPLE X Peptidyl Compounds TPI 792-33 and TPI 792-35 Kill Tumor Cells

This example demonstrates an assay for determining potency ofderepressors of IAP-inhibited caspases in cell cultures. This examplealso demonstrates that TPI 792-33 and TPI 792-35 reduce the viability oftumor cells in culture.

The TPI 792-33 and TPI 792-35 compounds were assayed to determine theireffects on tumor cell viability. As shown in FIG. 12, TPI 792-33 and TPI792-35 are tetrapeptides composed of unnatural amino acids that differin their amino acid sequence at the third position. The TPI 792-33 andTPI 792-35 compounds both have L-3-(2-thienyl)-alanyl,L-(2-naphthyl)-alanyl, and L-(e-fluorenylmethyloxycarbonyl)-lysinemoieties at positions 1 (N-terminus), 2 and 4, respectively, but differat position 3 where TPI 792-33 has L-p-chloro-phenylalanyl and TPI792-35 has a D-(e-fluorenylmethyloxycarbonyl)-lysyl moiety.

Cells from the prostate cancer cell line, ALVA31 express XIAP, as wellas other IAP-family proteins. The in vivo effects of TPI 792-33 or TPI792-35, either individually or in combination with the cytotoxicanticancer drug VP-16 (etoposide), on derepression of XIAP-inhibitedcaspase and viability of ALVA31 cells was determined as follows. ALVA31prostate cancer cells were seeded onto 96 well plates (10⁴ cells/well)in 100 μL RPMI containing 2.5% fetal bovine serum (FBS). After 24 hours,the IAP antagonists TPI 792-33, TPI 792-35 or the SMAC AVPI tetrapeptide(SEQ ID NO:4) was added at a final concentration of 40 μM with orwithout VP-16 (100 μM final concentration). After another 24 hrsincubation, cell viability was measured by the XTT dye-reduction assay(Roche, Molecular Biochemicals; Indianapolis, Ind.) and trypan blue dyeexclusion assay.

Anti-cancer drug VP-16 (etoposide), when administered alone to ALVA31cells, had essentially no effect on the viability of the cells in theXTT dye-reduction assay (FIG. 13) and trypan blue dye exclusion assay.The SMAC AVPI tetrapeptide (SEQ ID NO:4), when administered alone to theALVA31 cells, also had no effect on cell viability. In contrast, the TPI792-33 and TPI 792-35 peptides reduced viability of these prostatecancer cells by nearly half. Moreover, the combination of VP-16 withthese peptides resulted in more potent tumor cell killing compared toVP-16 alone. By comparison, the SMAC peptide was inactive, failing tosignificantly reduce the relative number of viable tumor cells under thesame culture conditions.

These results demonstrate that TPI 792-33 and TPI 792-35 displaymarkedly improved cellular activity compared to wild-type AVPI peptidefrom SMAC (SEQ ID NO:4). Furthermore, these results indicate that TPI792-33 and TPI 792-35 have the effect of increasing apoptosis in tumorcells by derepressing IAP-inhibited caspase. These results alsodemonstrate that TPI 792-33 and TPI 792-35 sensitize prostrate cancercells to the anticancer drug VP-16.

EXAMPLE XI Non-Peptidyl Compounds TPI 1396-34 and TPI 1391-28 Kill TumorCells

This example demonstrates that phenyl urea compounds (also calledpolyphenylurea compounds) identified from the TPI 1396 library anddiketopiperazine compounds identified from the TPI 1391 library reducethe viability of tumor cells in culture. This example furtherdemonstrates that cell killing activity for TPI 1396-34 and TPI 1391-28is specific for tumor cells.

The following assay was used to test the ability of individual compoundsfrom the TPI 1396 and TPI 1391 libraries to induce apoptosis of culturedtumor cell lines. Each of the compounds listed in Table IX wasindividually added to Jurkat leukemia cells (6.25×10⁵ cells/mL) in RPMIcontaining 2.5% FBS at various concentrations for 20 hours. Afterincubation, cells were washed and stained with FITC-conjugated Annexin Vantibody and propidium iodide (Biovision; Mountain View, Calif.). Cellswere incubated for 20 minutes at room temperature in the dark andfluorescence was measured by flow cytometry (FACScan, Immunocytometrysystem; Becton-Dickinson; San Jose, Calif.). Cells staining positive forAnnexin V were deemed non-viable.

As shown in Table IX, these compounds were able to induce cell death ina concentration dependent manner. Although SMAC was able to reduce cellviability by about 16% when present at 50 μM, several TPI 1396-34 andTPI 1391-28 compounds reduce cell viability by about 85 to 94%. Thus,compounds identified from the TPI 1396-34 and TPI 1391-28 libraries wereabout 5 to 6 fold more potent than SMAC at inducing apoptosis in tumorcells. A representative experiment testing additional compounds is shownin FIG. 27 a. TABLE IX Concentration μM 100 50 25 10 5 5 1 TPI 1391 %nonviable cells 1391-28 91 87 55 12 28 19 1391-21 94 91 44 11 18 161391-25 87 90 60 22 49 16 1391-17 91 88 45 N.T. 25 13 1391-5 88 88 36N.T. 17 1391-1 91 69 20 N.T. 18 1391-4 86 90 48 12 18 20 TPI 1396 %nonviable cells 1396-34 85 83 62 73 51 13 1396-12 85 89 89 95 95 151396-11 90 90 90 97 95 14 1396-28 13 14 13 13 SMAC 15 16 16 12

The TPI 1396-34 and TPI 1391-28 compounds were further tested as setforth below. FIG. 14 (Panel B) shows the structures for phenyl urea TPI1396-34 and diketopiperazine TPI 1391-28. Both of these compounds wereshown to induce apoptosis of cultured tumor cell lines in aconcentration-dependent manner using the assay described above, exceptthat compounds were added in the range of 0 to 20 μM.

As shown in FIG. 15, TPI 1396-34 and TPI 1391-28 killed Jurkat leukemiacells with EC₅₀ values of about 6.5 μM following a one-day exposure.Control compounds having the same core pharmacophore structure but withdifferent substituents at the R group which prevent binding to XIAP, didnot significantly reduce the viability of Jurkat leukemia cells underthe assay conditions.

Comparison of cell killing by TPI 1396-34 and TPI 1391-28 to therespective control compounds is shown in FIG. 16. One day treatment ofJurkat leukemia cells with 5 μM or 8 μM of TPI 1396-34, killed about 75%and 85% of cells, respectively. One day treatment of Jurkat leukemiacells with 5 μM or 8 μM of TPI 1391-28, killed about 45% and 80% ofcells, respectively. In contrast, the control compounds had nosignificant effect on the viability of these leukemia cells compared tountreated cells, indicating that the cytotoxic activity of thesecompounds is specific. Under the same assay conditions, 5 or 8 μM of theSMAC AVPI tetrapeptide (SEQ ID NO:4) had no significant effect on theviability of these leukemia cells, confirming that TPI 1396-34 and TPI1391-28 had far greater potency than SMAC. FIG. 27 b shows arepresentative assay as described above where Jurkat cells were culturedwith 10 μM 1396-34 for various times before measuring percent cell deathby annexin-V staining. As can be seen in FIG. 27 b, the kinetics ofapoptosis induction of Jurkat cells by TPI 1396-34 was rapid, withhalf-maximal killing achieved at approximately 12 hours and maximumkilling at about 24 hours.

Jurkat cells were also cultured with TPI 1396-34, a structurally relatedcompound TPI 1396-28, or SMAC 4-mer peptide AVPI (SEQ ID NO: 4) at finalconcentrations of 8 μM for 20 hours. After incubation, caspase-3 andcaspase-7 activity was measured in whole cells using a cell permeablesubstrate. As expected, TPI 1396-34 induced caspase activation, whileTPI 1396-28 (which differs from TPI 1396-34 only at R2) did not inducecaspase activation (see FIG. 27 c).

In addition, Jurkat cells were cultured with various concentrations ofTPI 1396-34 with or without 100 μM zVAD-fmk, which is a broad spectrumcaspase inhibitor. The percentage of cell death was measured 20 hourslater by annexin-V staining. Apoptosis induced by TPI 1396-34 wassuppressible by co-culturing the cells with zVAD-fink, (see FIG. 27 d).

A comparison of the effects of TPI 1396-34 on normal bone marrow cellsversus Jurkat leukemia cells was performed as follows. TPI 1396-34 (5μM) was incubated with Jurkat cells or normal bone marrow mononuclearcells (6.25×10⁵/mL) in RPMI and 2.5% FBS for 20 hours. After incubation,cells were washed, stained with FITC-conjugated Annexin V antibody andpropidium iodide, and fluorescence measured by flow cytometry asdescribed above.

As shown in FIG. 17, TPI 1396-34 and TPI 1391-28 caused little toxicityto normal bone marrow cells under the same culture conditions whererobust killing of the leukemia cells was observed. These resultsdemonstrate that the TPI 1396-34 and TPI 1391-28 selectively kill tumorcells compared to normal cells.

EXAMPLE XII Killing of Tumor Cells by TPI 1396-34 is Mediated by XIAP

This Example describes the effects of over-expressing wild type andmutant XIAP on tumor cell killing by TPI 1396-34.

U937 leukemia cells (6.25×10⁵ cells/mL) that had been stably transfectedwith either a Neo-control plasmid (U937-Neo cells) or a plasmid encodingXIAP (U937-XIAP cells) were treated with 5 or 8 μM of TPI 1396-34 inRPMI and 2.5% FBS for 20 hours. After incubation, cells were washed,stained with FITC-conjugated Annexin V antibody and propidium iodide,and fluorescence measured by flow cytometry as described in Example XI.

As shown in FIG. 18 and FIG. 27 e, over-expression of XIAP rendered U937cells resistant to TPI 1396-34. Comparisons of the effects of TPI1396-34 on U937-Neo and U937-XIAP cells demonstrated thatover-expression of XIAP correlated with resistance to apoptosisinduction by this agent. The increased resistance of tumor cells to theapoptogenic effects of TPI 1396-34 when the cells over-express XIAPindicates that TPI 1396-34 induces apoptosis by binding to XIAP.

Over-expression of XIAP in the U937-XIAP cells compared to vectortransfected control cells was confirmed by immunoblotting (FIG. 18,upper right panel). Expression of XIAP in K562 cells was included as acontrol, as these cells are known to express XIAP endogenously. Equalamounts of protein were subjected to SDS-PAGE (4-20% gradient gels fromISC BioExpress, Kaysville, Utah), followed by transfer to nitrocellulosemembranes. Membranes were probed with monoclonal mouse-anti human XIAP(0.25 mg/mL) (Transduction Laboratories, Lexington, Ky.) or monoclonalmouse-anti b-actin (1:3000 v/v) (Sigma Inc, Milwaukee, Wis.). Secondaryantibodies consisted of horseradish peroxidase (HRP)-conjugated goatanti-mouse IgG (Bio-Rad, Hercules, Calif.). Detection was performed bythe enhanced chemiluminescence (ECL) method. In FIG. 27 e, U937 cellsstably over-expressing XIAP or neomycin control transfectants werecultured with various concentrations of TPI 1396-34 for 20 hours and thepercentage of cell death was measured by annexin-V staining. Lysateswere prepared from the cells, normalized for total protein content andanalyzed by SDS-PAGE immunoblotting using antibodies specific for XIAP,caspase-3, and β-actin.

In addition to transfecting the cells with full-length XIAP, analogousassays were performed with HeLa cells transfected with plasmidsover-expressing various XIAP mutants. HeLa cells were transientlytransfected with plasmids encoding full-length, wild-type XIAP versusdeletion mutants having only the BIR2 (caspase-3/7 suppressing) domain,BIR3 (caspase-9 suppressing) domain, or a mutant in which both of theputative SMAC-binding pockets in BIR2 and BIR3 had been mutated to nolonger bind caspases. The mutant was produced by site-directedmutagenesis to modify positions 148, 219, 223, 314 and 323 to containalanine. HeLa cells were also transiently transfected with plasmidsencoding Bcl-XL, an anti-apoptotic protein that operates upstream ofcaspases to suppress Cytochrome C release from mitochondria. HeLa cells(2.5×10⁵) were seeded onto six well plates in 2 mL DMEM H21 with 5% FBS.After 24 hrs, cells were transfected (Gene Porter) with plasmids. At 48hrs after transfection, cells were treated with 5 μM of TPI 1396-34 for20 hours. Both floating and adherent cells were then recovered fromcultures, washed, and apoptosis was determined by Annexin V stainingusing flow cytometry.

As shown in FIG. 19, TPI 1396-34 induced apoptosis in HeLa cellstransfected with a control vector. Apoptosis induced by TPI 1396-34 wasnot blocked by over-expressing Bcl-XL, consistent with the fact thatBcl-XL operates upstream of XIAP. In contrast, HeLa cellsover-expressing full-length XIAP were protected from TPI 1396-34. Inaddition, cells expressing a mutant of XIAP in which the SMAC-bindingpocket of XIAP was mutated were not protected from the chemical compoundnor were cells expressing a mutant comprised of only the BIR2 domain.Cells expressing the BIR3 domain were protected from the apoptogenicactivity of TPI-1396-34. Taken together, these results indicate that TPI1396-34 induces apoptosis of tumor cell lines in culture by targetingXIAP.

As shown in FIG. 27 f, HeLa cells were transfected with plasmidsencoding XIAP, Bcl-X, CrmA, or empty vector. At 2 days aftertransfection, cells were treated with TPI 1396-34 (5 μM) for 20 hoursand the percentage of dead cells was measured by annexin-V staining.Consistent with IAPs representing a target of the polyphenylureacompounds disclosed herein, transient or stable over-expression of XIAPrendered tumor cell lines more resistant to apoptosis induction byactive compound, shifting the dose-response curve to the right, so thathigher concentrations of compound were required (FIGS. 27 e and 27 f).In contrast, over-expressing anti-apoptotic proteins Bcl-XL or CrmA didnot alter sensitivity of tumor cell lines to TPI 1396-34, demonstratinga specific effect (see FIG. 27 f). Bcl-XL over-expression did affordresistance to traditional anticancer drugs such as etoposide and CrmA (acaspase-8 inhibitor), and protected cells from apoptosis induced byTRAIL, confirming that these anti-apoptotic proteins were functional inthese experiments.

EXAMPLE XIII Broad Activity of Polyphenylurea Compounds AgainstTransformed Cells

This Example shows polyphenylurea compounds have activity against manydifferent tumor cell lines, while having little effect on normal cells.

Selected polyphenylurea compounds were tested on the National CancerInstitute (NCI) panel of 60 tumor cell lines (see FIG. 28 a and FIG.29). Cells were cultured with compounds for 48 hours followed bymeasurement of the relative number of viable cells using a protein-basedcolorimetric assay, expressing data as percent growth relative to cellstreated with solvent control alone. Compounds TPI 1396-11, TPI 1396-12,TPI 1396-22, and TPI 1396-34 induced reductions in viable cell numbers,with an average LD₅₀ (concentration required to kill 50% of the cells,after adjustment for background cell death) for the 60 cell lines of10+/−2.8 μM (median=17 μM), 7.6+/−12 μM (median=6 μM), 11+/−2.6 μM(median=22 μM), and 22+/−5 μM (median=23 μM), respectively. Moreover,the LD₅₀ was <10 μM for over one-third of the tumor cells treated withthe active compounds. In contrast, LD₅₀ was not reached for any of the60 tumor cell lines after treatment with up to 70 μM of the structurallyrelated control compound TPI 1396-28 (see FIG. 28 a). By comparison,when using this same assay, the mean LD₅₀ for the anticancer drugetoposide in the NCI panel of 60 tumor cell lines is 200+/−2.5 μM, withnone of the cells having LD₅₀<10 μM.

Compared to tumor cell lines, normal cells were relatively resistant tothe polyphenylurea compounds. As shown in FIG. 28 b, various types ofcells including Jurkat and HeLa tumor cell lines, and normal peripheralblood lymphocytes (PBLs), bone marrow mononuclear cells, mouse embryofibroblasts (MEFs), or human prostate epithelial cells were culturedwith various concentrations of TPI 1396-34. After 2 days, cell viabilitywas assessed by annexin-V staining.

When normal cells such as mouse embryo fibroblasts (MEFs), humanprostate epithelial cells, and peripheral blood lymphocytes (PBLs) werecultured with various concentrations of active poly-phenylurea compoundTPI 1396-34, the slope of the cell cytotoxicity curve was much flatterthan observed for tumor cell lines (FIG. 28 b). At a concentration of 10mM, for example, the percentage cell death increased by less thanone-fold above background for these types of normal cells, while killingof tumor lines such as Jurkat and HeLa increased by >4 fold. Activatinglymphocytes with the mitogenic lectin, phytohemaglutinin (PHA), did notincrease sensitivity to the XIAP-antagonists. Normal human bone marrowmononuclear cells (BM) tended to be more sensitive. However, even forthese cells, the LD₅₀ was not reached at concentrations up to 40 mM. Bycomparison, the LD₅₀ of Jurkat and HeLa cells was achieved atconcentrations of about 5 μM (FIG. 28 b).

Since many tumor and leukemia cell lines proliferate faster than normalcells, it was investigated whether the polyphenylurea compounds couldinduce apoptosis of non-replicating malignant cells. Accordingly,freshly isolated chronic lymphocytic leukemia (CLL) B-cells from fivepatients and freshly isolated leukemic blasts from five patients withacute myelogenous leukemia (AML) were treated for 20-24 hours in vitrowith compounds TPI 1396-11, TPI 1396-12, or TPI 1396-34 versus TPI1396-28 control compound or AVPI peptide (SEQ ID NO: 4), and thepercentage of cell death was measured by annexin-V/propidium iodidestaining with FACS analysis (FIG. 28 c) or annexin-V staining (FIG. 28d). These leukemic cell samples contained only small percentages ofcycling cells, and did not replicate under standard culture conditions.As seen in FIGS. 28 c and 28 d, active polyphenylurea compounds induceddose-dependent cell death of primary-cultured leukemia cells in 5 of 5CLL specimens (FIG. 28 c) and 4 of 5 AML specimens (FIG. 28 d) examined,with LD₅₀ achieved at doses of approximately 5 μM after correction forspontaneous apoptosis in culture. In contrast, the inactive controlcompound TPI 1396-28 and the AVPI peptide did not induce apoptosis ofthese leukemia cells. All samples were treated with both TPI 1396-34,TPI 1396-28, as well as the AVPI peptide, but the complete data set isshown only for AML-1. Comparable results were obtained with controlcompounds for the other leukemia specimens. In addition, the activepolyphenylurea compounds were also active against transformedhematopoietic cells from mice, inducing death of mouse 70Z/3 lymphomaand immortalized 32D myeloid cells with EC₅₀ valves of 8 to 12 μM. Thus,cell replication is not required for sensitivity to the disclosedpoly-phenylurea compounds.

EXAMPLE XIV Polyphenylurea Compounds Sensitize Tumor Cells to AnticancerDrugs and TRAIL

This example shows that polyphenylurea compounds can collaborate withconventional anticancer drugs to induce killing of tumor cells.

In order to test the effect of polyphenylurea compounds in combinationwith known anticancer drugs, Du145 prostate cancer cells were culturedfor 48 hours with various concentrations of Etoposide (VP16),Doxorubicin (DOX), or Paclitaxel (TAXOL) with or without 10 μM TPI1396-34. The percentage of viable cells relative to control wasdetermined using a MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assaywhich is commercially available (Sigma). FIG. 30 a shows representativedata indicating that TPI 1396-34 significantly increased dose-dependentcytotoxicity of VP16, DOX, and TAXOL in Du145 prostate cancer cells. Amore complete version of the data showing results at variousconcentrations of TPI 1396-34 is shown in FIG. 31 a. Similar resultswere obtained for PPC1 (FIG. 31 b) and PC3 (FIG. 31 c) prostate cancercells treated with VP16, DOX, or TAXOL, and for H460 (FIG. 31 d) lungcancer cells treated with VP16 or DOX. Inactive poly-phenylureascompounds failed to sensitize tumor cells to anticancer drugs.

Similar tests of the effects of polyphenyl urea compounds on apoptosisinduction by the biological agent TRAIL, an apoptosis inducing member ofthe Tumor Necrosis Factor (TNF) family were also performed. Cancer celllines PPC1, ALVA31 and DU145 were treated with various concentrations ofTRAIL alone or in combination with TPI 1396-34 at a concentration of 1μM. TPI 1396-34 sensitized PPC1, ALVA31, DU145, and HeLa cells toTRAIL-induced apoptosis (see FIG. 30 b). Inactive control compounds didnot display this activity.

EXAMPLE XV Polyphenylurea Compounds Demonstrate Anti-Tumor Activity inClonogenic Survival Assays and Tumor Xenograft Models

This example shows that clonogenic survival of cancer cells is reducedby polyphenyl urea compounds. In addition, selected polyphenylureacompounds showed anti-tumor activity in vivo using human tumorxenografts grown in immunocompromised mice.

In addition to short-term cytotoxicity assays, selected polyphenylureacompounds were tested for effects on clonogenic survival of cancer cellsin colony formation assays, which can be considered a more stringenttest of anticancer activity. Two prostate cancer cell lines PC-3 andLNCaP were cultured with TPI 1396-34 for 3 days, then culture medium waschanged and colonies were counted one week later. FIG. 32 a shows theresults obtained using various concentrations of TPI 1396-34 and oneconcentration (10 μM) of a control compound. As seen in FIG. 32 a, TPI1396-34 diminished clonogenic survival of these cancer lines in aconcentration dependent manner, with an average EC₅₀ dose of 3 μM+0.5 μM(mean+std error). At a dose of 10 μM, colony formation was reduced to<5% of control, in contrast to inactive control compounds, which hadrelatively little effect.

Selected polyphenylurea compounds were tested for anti-tumor activity invivo, using human tumor xenografts grown in immunocompromised mice.First, it was determined what doses of polyphenylurea compounds weretolerated by mice. It was found that 100 mg/kg delivered i.p. as asingle or in divided doses resulted in no gross toxicity. For tumorxenograft studies, PPC1 prostate cancer cells (2.5 million) wereinjected subcutaneously into the flanks of 8 male Balb/C nu−/nu−mice.Half of the animals received i.p. injections of TPI 1396-34 in DMF(N,N-Dimethylformamide) at 30 mg/kg at day 7 and day 8, while the otherhalf received DMF diluent alone. Tumor growth was monitored at leasttwice weekly by external calipers (see FIG. 32 b). At 24 days aftercompound injections, mice were sacrificed and tumors were excised andweighed (see FIG. 32 b, inset). As can be seen in FIG. 32 b, treatmentwith TPI 1396-34 resulted in reduced tumor size compared to the DMFcontrol.

Similar data was obtained using TPI 1396-22 (see FIGS. 33 a and 33 b).In these experiments PPC1 prostate cancer cells (2.5 million) wereinjected subcutaneously into the flanks of male Balb/C nu−/nu− mice. Ondays 5, 6, and 7, when tumors were about 125 mm³, mice were treated with30 mg/kg of TPI 1396-22 or solvent by i.p. injection and tumor volumewas measured by calipers at least twice weekly for 19 days afterinjection. In FIG. 33 b, the mice were sacrificed at day 19 and thetumors excised and weighed.

Additional experiments were performed using a different tumor xenograftmodel. HCT-116 colon cancer cells (2.5 million) were injectedsubcutaneously in the flanks of female Balb/C nu−/nu− mice. On days 6,7, and 8 when tumors were about 125 mm³, mice were treated with 30 mg/kgof TPI 1396-34 (n=10) or solvent control (n=19) by i.p. injection. Tumorvolume was measured by external calipers at least twice weekly for 19days (see FIG. 32 c). On day 19, the mice were sacrificed and the tumorswere excised and weighed. Again, tumor size was reduced in mice treatedwith TPI 1396-34 compared to solvent control.

In summary, dosing mice for just 2 or 3 days with TPI 1396-34 or TPI1396-22 significantly slowed the rate of growth of both PPC1 and HCT116tumors, thus demonstrating in vivo anti-tumor activity of these chemicalcompounds.

EXAMPLE XVI Structure Activity Relationship (SAR) of Individual TPI 1396Compounds and the Generation of TPI 1509 Compounds

This Example shows SAR information based on TPI 1396-1 through TPI1396-36 (see FIG. 22 for structures of these compounds) and thestructure of TPI 1509 compounds. TPI 1509 compounds are based on TPI1396 compounds which are in turn based on the TPI 927 library. As shownby the activity screening of individual compounds TPI 1396-1 through TPI1396-36, a number of different hydrophobic aromatic groups areacceptable for activity at the R1 and R3 positions (see Table X). TableX also shows that active compounds can be derived from a proline at theR2 position. TABLE X Core structure with R2 = L-Proline

TPI 1396# R1 R3 Activity* 10

2.8 11

2.6 12

2.7 22

3.3 23

2.6 34

3.0 35

2.5*Relative caspase-3 activity in the XIAP derepression assay wascalculated as the ratio of the Vmax in the presence of each compounddivided by the Vmax of the controls having caspase-3 and XIAP.

When examining individual compounds having different functionalities atthe R2 position (Table XI), it can be seen that the diphenyl ureasderived from triamines having two secondary amines and one tertiaryamine are more active than the triphenyl ureas. These diphenyl ureaswere derived from the reduction of proline-containing acylated dipeptideamides, followed by treatment with phenyl isocyanate.

TPI 1396- Structure Activity* 34

3.0 25

1.6 28

1.3 31

1.3*Relative caspase-3 activity in the XIAP derepression assay wascalculated as the ratio of the Vmax in the presence of each compounddivided by the Vmax of the controls having caspase 3 and XIAP.

Also, a series of compounds (TPI 1509, shown in FIG. 34) synthesizedusing D-proline instead of L-proline were tested for activity. FIG. 34shows the names, structures and activity of these compounds in the XIAPderepression assay. All of these compounds were active at 25 mg/ml, andthe most active compounds in this assay were TPI 1509-1, TPI 1509-2, TPI1509-3, and TPI 1509-6.

EXAMPLE XVII Structure Activity Relationship (SAR) of Polyphenylureas

This example demonstrates compounds that can be used to address therelative importance of the main scaffold and each R group on theactivity of the poly-phenylurea compounds. FIG. 35 shows compounds whichare analogs of TPI 1509-7 where the properties of R1, R2 and R3 arevaried separately by altering their chemical natures and therefore theirphysiochemical properties. A similar series of compounds was synthesizedbased on TPI 1396-34. The compounds are assayed for activity and anotherround of SAR can optionally be performed to further optimize thestructure of the compounds. A desirable compound can be, for example, acompound with a lower molecule weight and better pharmacologicalproperties than existing compounds.

EXAMPLE XVIII TPI 1332 Peptide Compounds Interact with a Site on XIAPDistinct from the SMAC Binding Site

This example demonstrates that compounds in the TPI 1332 series oftetrapeptides do not interact with the SMAC binding site on XIAP.

Peptide compounds containing unnatural amino acids in the 792 series,792-33 and 792-35 (see FIGS. 12 and 20 and Example XI), and an activecompound in the TPI 1332 series, TPI 1332-69, were tested for bindingactivity in the SMAC binding assay as described above in Example X. Forthis assay, biotinylated SMAC 7-mer peptide (50 ng) was bound to 96 wellplates coated with NeutrAvidin (Pierce, Rockford, Ill.) at 1 mg/ml in100 μl per well of 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM EDTA, 10%sucrose, 0.1% CHAPS, 10 mM DTT. Then GST-XIAP was added at 0.1 mg/ml in100 μl with or without compounds in DMSO. After incubation for 1 hour atroom temperature, plates were washed with PBS with 0.05% Tween 20, andbound GST-XIAP was detected by addition of mouse anti-GST monoclonalantibody (1:2000 dilution) followed by anti-mouse horse radishperoxidase conjugated IgG and 3,3′,5,5′-tetramethylbenzidine base (TMB)substrate with detection at 450 nm on a plate reader.

As shown in FIGS. 36 A-F and 37, the active compounds of the TPI 792series and its analogs, as well as compounds of the TPI 1332 series,compete with XIAP binding to the SMAC peptide, with the exception of TPI1332-69. In addition, as shown in FIG. 37, while TPI 1332-69 is activein the derepression assay but does not compete for the SMAC peptidebinding site, TPI 1495-5 (substitution analog of TPI 1332-69 with G atposition 4) is active in the derepression assay and competes for theSMAC peptide binding site. As expected, the inactive compounds in the792 or 1332 series do not compete with XIAP binding to the SMAC peptide.

As is shown in FIG. 36C-F, many compounds of the 1332 series werecapable of reversing XIAP-mediated suppression of caspase 3. Activecompounds at 50 μg/ml (FIG. 36C) included TPI 1332-1, -3, -4, -5, -11,-15, -32, -36, -38, -40, -41, -42, -45, -47, -63 to -69, -71 to -73,-76, -78, -81 to -85, -87 to -90 and -93. The activity of TPI 1332-1,-4, -41, -53, -69, and -77 was also determined in the derepression assayusing XIAP-BIR2 domain, as shown in FIG. 36E. The activity of TPI1332-1, -2, -4, -6, -41, -47, -53, -55, -69, -76, -77 and -85, in thederepression assay using the cIAP1 BIR2 domain further is shown in FIG.36F. These data indicate that TPI 1332 and related compounds are activein derepressing caspase inhibited by XIAP or the BIR2 domain of XIAP,but do not overcome cIAP1-mediated suppression of caspase-3. It isimportant to note that the lack of activity observed for variouscompounds can be the result of the compounds being present at a two foldexcess over cIAP1.

Shown in FIG. 37 are the activities of compounds derived from TPI1332-69, referred to as TPI 1495-1 (TPI 1332-69) and TPI 1495-2 throughTPI 1495-9. Whereas TPI 1332-69 was active in the caspase derepressionassay but inactive in the SMAC competition assay, TPI 1495-5 was activein both the caspase derepression assay and the SMAC competition assay.These data indicate that TPI 1495-5 has a binding site on XIAP thataffects the functions of both the BIR2 domain and SMAC binding domain ofXIAP.

FIG. 43 shows several compounds derived from TPI 792-33 or TPI 792-35,referred to as the TPI 1453 series. As is shown, TPI 1453-1 is the sameas TPI 792-33, with TPI 1453-2 through TPI 1453-5 being aremodifications of TPI 792-33 that contain various natural and nonnaturalamino acid substitutions; TPI 1453-5 is the same as TPI 792-35, with TPI1453-6 through TPI 1453-9 being are modifications of TPI 792-35 thatcontain various natural and nonnatural amino acid substitutions.Compounds TPI 1453-1, -2, -4, -6, -7, -8, and -9 were determined to haveactivity in both the caspase derepression assay and the SMAC competitionassay.

These data indicate that a novel negative regulatory site on XIAP, theXIAP BIR2 domain, is not targeted by SMAC. Compounds that bind to thisnovel negative regulatory site such as TPI 1332-36, as well as compoundsthat modulate both the SMAC binding site and BIR2 domain, can be used inscreening assay in order to identify other compounds that can bind tothis novel site.

EXAMPLE XIX Identification of Compounds that Inhibit IAPs Other thanXIAP

This example describes an assay that can be used to determine theeffects of derepressors of XIAP-inhibited caspases on other IAPs.

Immunohistochemical analysis of prostate cancers indicates that cIAP1and cIAP2 are commonly over-expressed in these tumors. Both cIAP1 andcIAP2 are caspase inhibitors (Roy, EMBO J. 16:6914-6925 (1997)) and theyeach bind SMAC (Du et al., Cell 102:33-42 (2000); Chai et al., Nature406:855-862 (2000)). Moreover, molecular modeling studies indicate thatsome of the BIRs of cIAP1 and cIAP2 are likely to bind SMAC, havinggreat structural similarity to XIAP. These observations indicate thatderepressors of XIAP-inhibited caspases can have activity againstcaspases inhibited by these other IAPs.

To confirm that derepressors of XIAP-inhibited caspases have activityagainst caspases inhibited by these other IAPs the following assays areperformed. Competition of the compounds with the SMAC peptide forbinding to BIRs on XIAP is assayed. To accomplish this, the compoundsare tested in SMAC competition assays in which FITC-conjugated SMACtetrapeptide AVPI (SEQ ID NO:4) or FITC-conjugated HtrA2 tetra-peptideAVPS (SEQ ID NO:6) are bound to BIRs from XIAP. Rather than expressingfull-length XIAP, fragments of XIAP containing only the BIR2 or BIR3domains are expressed, as described in Takahashi et al., J. Biol. Chem.273:7787-7790 (1998) and Deveraux et al., EMBO J. 17:2215-2223 (1998).These assays will determine if the compound functions as a SMAC-mimic,and also whether the compound targets BIR2 (the domain that inhibitscaspases-3 and -7), BIR3 (the domain that inhibits caspase-9), both, orneither of these domains.

Additionally, enzyme depression assays are performed using BIR2 or BIR3domains to pinpoint the domain in XIAP that is targeted by a compound.Recombinant purified BIR2 is mixed with caspase-3, and BIR3 withcaspase-9, then the activity of these proteases is measured againstspecific fluorogenic substrate peptides (Ac-DEVD-AFC for caspase-3versus Ac-LEHD-AFC for caspase-9) in the presence and absence of acompound in an effort to pinpoint whether the compound targets BIR2,BIR3, both or neither of these domains in the XIAP protein. Theseresults can be used for structure-based optimization of compounds usingmolecular modeling of the published structures of XIAP, BIR2 (Sun etal., Nature 401:818-821 (1999) and Riedl et al., Cell 104:791-800(2001)), and BIR3 (Liu et al., Nature 408:1004-1008 (2000)).

With respect to cIAP1 and cIAP2, similar enzyme derepression and SMACcompetition assays are performed using full-length cIAP1 and cIAP2, aswell as fragments containing individual BIR domains, thus determiningwhether the compounds cross-inhibit these other members of theIAP-family.

If a compound does inhibit cIAP1, cIAP2, or both of these proteins, thenthe potency of the compound can be improved through medicinal andcombinatorial chemistry. Assays can be performed to contrast retentionversus loss of cIAP1/cIAP2 activity in vitro with activity of compoundsin cell-based assays. Structure activity relationship studies of thistype indicate whether the optimal compound has selective specificity forXIAP versus pan-reactivity against several IAPs. The compounds withthese different profiles (selective versus broad-spectrum activity) arecontrasted with respect to toxicity issues, to obtain a compound with adesired balance between efficacy and safety.

EXAMPLE XX Mechanism of Action of PolyPhenyurea Compounds

To determine whether apoptosis induction by polyphenylurea compoundsoccurs through the intended mechanism of action, toxicity of TPI 1396-34was tested using cells obtained from XIAP knock-out mice in a cell basedassay. Mechanism-based toxicity of TPI 1396-34 and daunorubicin on mouseembryo fibroblasts (MEFs) from XIAP −/− mice and transformed wild type(+/+) mice was determined. Cells were either tested directly at lowpassage (FIGS. 38 A and C) or after transformation by infection with aretrovirus encoding SV40 large T antigen (FIGS. 38 B and D). Cells werecultured 1 day with various concentrations of compound TPI 1396-34(FIGS. 38 A and B) or with daunorubicin (FIGS. 38 C and D). Cellviability was measured by MTT assay, expressing data as a percentagerelative to control, untreated cells. Data shown in FIG. 38A-D representmean±standard deviation of triplicate determinations.

These results demonstrate that XIAP-deficient cells are less sensitiveto the polyphenylurea compound compared to wild-type MEFs, providingevidence that the compound functions through the intended mechanism ofaction since cells lacking the intended target (XIAP protein) are lesssensitive. In contrast, if the compound induced apoptosis through anon-specific mechanism, it would be expected that XIAP-deficient cellswould be more sensitive due to the absence of this anti-apoptoticprotein. These findings also contrast non-transformed with transformedcells by showing that transformed cells are more sensitive to the XIAPantagonist. In contrast, conventional anticancer drugs such asdaunorubicin do not display selectivity for transformed cells in thesein vitro cytoxicity assays.

EXAMPLE XXI Polyphenylurea Compounds Enhance Cytotoxicity ofAntigen-Specific CTL

To determine if polyphenylurea compounds reduce resistance to apoptosismechanisms relevant to CTL-mediated cell lysis, selected compounds weretested for their ability to enhance cytotoxicity of antigen-specificCTL.

For these experiments, tumor cells were loaded with ⁵¹Cr then pulsedwith specific antigen and incubated with antigen-specific T cells ateffector:target ratios of either 5 (FIG. 39A) or 10 (FIG. 39B) in theabsence of compounds (open circles; dashed lines) or in the presence of10 μM of either inactive control compound TPI 1396-28 (squares) oractive compound TPI 1396-34 (closed circles). After 4 hours, ⁵¹Crrelease was measured. Data shown in FIG. 39 are expressed as apercentage of total release induced by detergent lysis, and datarepresent mean±standard deviation of duplicate determinations.

As is shown in FIG. 39, TPI 1396-34 sensitizes tumor targets toCTL-mediated lysis. These results provide evidence that polyphenylureacompounds are not deleterious to CTL effector function and indicate thatinhibiting XIAP reduces resistance to apoptosis mechanisms relevant toCTL-mediated cell lysis.

EXAMPLE XXII In Vivo Activation of Caspases by Polyphenylurea Compoundsin Tumors

To determine if polyphenylurea compounds induce caspase activation intumors in vivo, TPI 1396-12 was tested a human tumor xenograft mousemodel. For these studies, tumor-bearing Balb/c mice at 8 weeks of agewere either injected i.p. for 3 successive days with 30 mg/kg ofpolyphenylurea compound TPI 1396-12 or with an equal volume of diluent(CNTL). Immunoblot analyses of tumor tissue, the results of which areshown in FIG. 40A, were performed using an antibody specific for cleavedcaspase-3 or actin at 24 hours following the final injection of compoundor control. These results indicate that polyphenylurea compound TPI1396-12 induce caspase activation in tumors in vivo.

FIG. 40B shows immunohistochemistry of tumor tissue sections using H& Estained sections (B and C); anti-caspase-3 antibodies and anti-PCNAantibodies (dark stained nuclei; D and E), anti-caspase-6 antibodies(dark staining; F and G) and anti-DFF40 antibodies (dark staining; H andI). As is shown by detection of surrogate marker PCNA, polyphenylureacompound TPI 1396-12 had no effect on tumor proliferation. These resultsprovide further evidence that polyphenylurea compound TPI 1396-12functions through an apoptotic mechanism in vivo.

EXAMPLE XXIII In Vivo Toxicology Analysis of Polyphenylurea Compounds

To assess in vivo stability and toxicology of polyphenylurea compoundTPI 1396-12, tumor-bearing mice were treated with doses of compoundspreviously determined to be adequate for achieving anti-tumor activityin vivo using xenograft model and toxicological analyses were performed.In these studies, Balb/c mice (8 weeks of age) were either untreated orinjected i.p. for 3 successive days with 30 mg/kg of polyphenylureacompound TPI 1396-12, or with an equal volume of diluent (PBS containing10% DMSO, 5% TWEEN 80). At 12 hours after the final injection, mice weresacrificed and blood was analyzed for white blood cell count (WBC), redblood cell count (RBC), and platelet count (PLT). Sera were assayed forBUN, bilirubin, ALT and AST. These data, shown in FIG. 41, represent themean±standard deviation for 3 mice. Although these data do not reachstatistical significance, they indicate a trend.

Toxicology data shown in FIG. 41 indicate that polyphenylurea compoundTPI 1396-12 is not toxin at the administered dosage. In addition,histological analyses of tissues confirmed these results.

These and related studies indicated that polyphenylurea compound TPI1396-12 has a maximum tolerated dose of greater than 200-400 mg/kg inmice (non-lethal); that anti-tumor activity was demonstrated with aslittle as two sequential daily 30 mg/kg i.p. doses; and thatpolyphenylurea compound TPI 1396-12 is expected to be stable in humanserum for greater than 48 hours.

EXAMPLE XXIV Polyphenylurea Compounds Selectively Bind to BIR2

To obtain direct evidence that polyphenylurea compounds bind to the BIR2domain of XIAP, NMR studies were performed. In these studies,polyphenylurea compounds TPI 1540-14 and TPI 1540-15 were shown toselectively bind to BIR2.

T_(1r) measurements were formed at 200 ms with 400 μM polyphenylureacompound TPI 1540-14, -15 or -20 in the absence and presence of 10 μMGST-BIR2. Binding of active compounds TPI 1540-14 and TPI 1540-15 wasmanifested by a decrease in signal intensity in the presence of asub-stoichiometric amount of GST-BIR2. Inactive compound TPI 1540-14 didnot show this effect. As a control, an internal reference compound wasadded to the solution containing TPI 1540-15 (marked with a * in FIG.42). As a control for compound binding to GST, the binding of TPI1540-15 was also tested against GST-Bcl-B, which produced a negativeresult. Results for TPI 1540-14 and TPI 1540-20 are shown in FIG. 42.Results for TPI 1540-15 were similar to those observed for TPI 1540-14.

In summary, this example provides evidence that polyphenylurea compoundsTPI 1540-14 and TPI 1540-15 bind directly to the BIR2 domain of XIAP.

EXAMPLE XXV Structure Activity Relationship (SAR) of TPI 1540 Compounds

This example shows SAR information for individual TPI 1540 compounds TPI1540-6 through TPI 1540-23. As is shown in FIG. 35B, a number ofdifferent modifications can be made without altering activity incomparison to TPI 1509-7 or TPI 1396-34 as indicated by IC50 valuesobserved in the XIAP derepression assay or Jurkat-Annexin V assay.

These SAR data show that structures of active compounds, such as TPI1396-34 can be simplified while retaining complete activity against thetarget protein both in vitro and in cell based assays. In addition,these data show that polyphenylurea compounds can be optimized withrespect to Lipinsky's rules and that, accordingly, pharmacologicalproperties of selected polyphenylurea compounds can be optimized.

EXAMPLE XXVI Binding of BID and XIAP BIR2 Domain to BiotinylatedPeptides

This example describes competition assays for XIAP-BIR2 binding tobiotinylated peptides of the TPI 792, TPI 1332 and TPI 1495 series. Thebiotinylated peptides are referred to as the TPI 1554 series, andinclude TPI 1554-1 through TPI 1554-8, as shown in FIG. 44. Also shownin the table of FIG. 44 is the TPI number corresponding to the originaltetrapeptides (Non-biotin Synthesis #) and the molecular weights (MW).

Binding assays were performed to determine whether the biotinylatedpeptides bound to the BIR domain of XIAP. Biotinylated peptides wereadsorbed to Neutravidin-coated plates, and GST-XIAP BIR2 or GST-BID(control) was added. Bound GST polypeptides were detected with ananti-GST antibody. FIGS. 45A-J show that biotinylated compounds TPI1453-1 (TPI 1554-1) (A); TPI 1453-6 (TPI 1554-2) (B); TPI 1332-4 (TPI1554-3) (C); TPI 1332-41 (TPI 1554-4) (D); TPI 1332-69 (TPI 1554-5) (E);TPI 1332-77 (TPI 1554-6) (F); TPI 1495-19 (TPI 1554-7) (G), and TPI1495-20 (TPI 1554-8) (H) competed for binding to XIAP-BIR2 to an extentcomparable to the binding of SMAC peptides SMAC 7-mer (I) and SMAC 4-mer(J).

FIGS. 46 A-C show binding of XIAP-BIR2 to biotinylated tetrapeptides TPI1554-1 through TPI 1554-8 using 1 μg/ml XIAP BIR2 (A); 0.5 μg/ml XIAPBIR2 (B), and 0.25 μg/ml XIAP BIR2 (C) with biotinylated peptides atconcentrations of 1.6 μg/ml, 0.4 μg/ml and 0.1 μg/ml. TPI 1554-3 throughTPI 1554-8 had a greater extent of binding to XIAP BIR2 domain incomparison to TPI 1554-1 and -2. These results show that binding of XIAPBIR domain to biotinylated tetrapeptides TPI 1554-3 through TPI 1554-8is comparable to the binding of SMAC peptides.

FIG. 47 shows competition assays for the binding of XIAP BIR2 domain tothe biotinylated tetrapeptides in the absence or presence of apolyphenylurea compound, using biotinylated peptide TPI 1554-5 (TPI1332-69) (A), which is a non-SMAC mimic; and biotinylated peptide TPI1554-3 (TPI 1332-4) (B), which is a SMAC mimic. Results from thesestudies indicate that the polyphenylureas do not compete for binding ofXIAP-BIR2 domain with either of the tetrapeptides. However, the SMACmimic tetrapeptide TPI 1554-3 competes with binding of XIAP BIR2 domainto the biotinylated non-SMAC mimic tetrapeptide TPI 1554-5. Thisobservation for TPI 1554-3 is consistent with the finding describedherein in Example XVIII that TPI 1332-69 binds to a site on XIAP thatoverlaps between the SMAC and BIR2 binding sites.

EXAMPLE XXVII Rhodamine-Labeled Binding/Competitive Binding Assay

This Example describes assays useful for determining the bindingaffinity of a derepressor of an IAP-inhibited caspase for an IAP, orfunctional fragment thereof.

A. Binding Assay

A polarization-based binding assay was used to detect binding betweenrhodamine labeled candidate derepressors of IAP inhibited caspase(candidate compounds) and the XIAP fragments His-BIR2, His-BIR1-2 andHis-BIR1-2-3. The assay is based on the decrease in mobility that occursfor rhodamine-labeled candidate compounds when associated with XIAP orfunctional fragments thereof, which is detected as an increase inpolarization of the rhodamine-labeled candidate compound due to bindingto the target protein.

In a first assay, binding of candidate derepressors of IAP inhibitedcaspase was measured by a fluorescence polarization procedure. A fixedquantity of rhodamine-labeled candidate compound was titrated againstvarying quantities of IAP fragment compounds (His-BIR2 of XIAP,His-Traf2 (negative protein control), His-BIR1-2 of XIAP or His-BIR1-2-3of XIAP). Binding of the candidate compound to the IAP fragment isdetected as an increase in polarization (millipolars or mP) compared tothe candidate compound alone, as bound fluorophore has a greaterpolarizing effect than unbound fluorophore. A plot of mP versus logconcentration of the IAP fragment can be used to calculate the bindingconstant for the candidate compound for the IAP fragment.

The results of this assays are set forth in FIGS. 48, 50 and 52.Briefly, a micromolar quantity of labeled candidate compound wasprepared in a buffered solution in the presence of various amounts ofHis-BIR2 of XIAP, BIR1 of NAP, and His-BIR1-2 of XIAP in a standardmicrotiter plate. (See Table XII for conditions.) His-Traf2 served as anegative control for specificity. Results similar to those seen withHis-BIR1-2 of XIAP are seen with His-BIR1-2-3 of XIAP. The plates wereincubated for one hour at room temperature, after which polarization ofrhodamine was read in an LJL Analyst HT® multimode reader withexcitation at 530 nm and emission at 580 nm. FIGS. 48, 50 and 52 showpolarization values (millipolars, mP) plotted as a function ofmillipolars versus the log concentration of His-BIR2 of XIAP, His-Traf2or His-BIR1-2 of XIAP. His-Traf2 was plotted as a negative control.Results similar to those seen with His-BIR1-2 of XIAP are seen withHis-BIR1-2-3 of XIAP.

The structure of TPI 1332-4 is shown in FIG. 36A. The structure of TPI1540-14 is set forth in FIG. 35A. TABLE XII Candidate Compound(Rhodamine- Concentration of Labeled Rhodamine Labeled CandidateCandidate Compound IAP Fragment/ FIG. Compound) (in Buffer) [IAPFragment] μM 48 TPI 1332-4 2.4 μM His-BIR2 of XIAP, (TPI (in 50 mM KPi,pH 7.4, His-Traf2/ 1566-11) 50 mM NaCl) 0, 0.11, 0.33, 0.99, 2.96, 8.89,26.67 50 TPI 1540-14 2.5 μM His-Traf2, (TPI 1576 (in 50 mM Tris, pH 8.8,His-BIR1-2 of XIAP/ pk1, pk2) 50 mM NaCl, 1.25 mM 0, 0.14, 0.41, 1.23,DTT) 3.70, 11.11, 33.33, 100 52 TPI 1540-14 2.5 μM His-BIR1-2 of XIAP,(TPI 1576 (in 50 mM Tris, pH 8.8, His-Traf2, pk1, pk2) 50 mM NaCl, 1.25mM BIR1 of XIAP/ DTT) 0, 0.14, 041, 1.23, 3.70, 11.11, 33.33, 100

The structure of TPI 1332-4 is shown in FIG. 36A. The structure ofrhodamine-labeled TPI 1332-4 (TPI 1566-11) is shown below.

The structure of TPI 1540-14 is set forth in FIG. 35A. The structures oftwo species of rhodamine-labeled TPI 1540-14 (TPI 1576-37 pk1 and TPI1576-37 pk2) are shown below.

B. Competitive Binding Assay

In a second assay, competitive binding of candidate derepressors of IAPinhibited caspase was measured by a fluorescence polarization procedure.Fixed quantities of rhodamine-labeled candidate compound and IAPfragment were titrated against varying concentrations of a knownIAP-binding compound. Displacement of the candidate compound by theknown IAP-binding compound was detected as a decrease in polarization,as the displaced (unbound) candidate compound will have a lowerpolarization than the bound candidate compound. Polarization(millipolars or mP) was plotted against log concentration (log [ ]) ofthe known IAP-binding compound. A plot of mP versus log concentration ofthe IAP-binding compound was then used to calculate the binding constantfor the candidate compound-the IAP fragment pair.

The results of this assays are set forth in FIGS. 49, 51 and 53.Briefly, a micromolar quantity of labeled candidate compound and IAPfragment was prepared in a buffered solution in the presence of variousamounts of IAP-binding compound TPI 1396-11 in a standard 96-wellmicrotiter plate. (See Table XIII for conditions). Plates were incubatedfor one hour at room temperature, after which polarization of rhodaminewas read in an LJL Analyst HT® multimode reader with excitation at 530nm and emission at 580 nm. FIGS. 49, 51 and 53 show polarization values(millipolars, mP) plotted as a function of the log concentration of TPI1396-11 in μg/ml. FIGS. 49 and 53 also show the IC₅₀ of the labeledcandidate compounds (10 μM for TPI 1332-4, 36 μM for TPI 1540-14). TABLEXIII Labeled Concentration of Candidate Rhodamine Labeled CompoundCandidate Compound Figure (I.C.₅₀) (in Buffer) [TPI 1396-11] μg/mL 49TPI 1566-11 2.4 μM 0, 1.56, 3.13, 6.25, (10 μM) (in 50 mM KPi, pH 7.4,12.5, 25, 50, 100 50 mM NaCl) 51 TPI 1576-37 2.5 μM 0, 1.56, 3.13, 6.25,pk2 (50 mM Tris, pH 8.8, 50 12.5, 25, 50, 100 mM NaCl, 1.25mM DTT) 53TPI 1576-41 2.5 μM 0, 1.56, 3.13, 6.25, pk2 (56 μM) (50 mM Tris, pH 8.8,50 12.5, 25, 50, 100 mM NaCl, 1.25 mM DTT)

EXAMPLE XXVIII Structure Activity Relationship (SAR) of Individual TPI1577, TPI 1567 and TPI 1572

This Example shows the structure and SAR information based on TPI 1577,TPI 1567 and TPI 1572, which are all based on TPI 1540-14. As shown bythe activity screening of individual compounds belonging to the TPI1577, TPI 1567 and TPI 1572 families, a number of different hydrophobicgroups are acceptable for activity at the R1, R3, N″, positions (seeTable XIV). TABLE XIV TPI # — MW¹ — Activity² MLogP Structure (μM) TPI1577-1 —425 —2.71

252 TPI 1577-2 —467 —3.31

157 TPI 1577-3 —411 —2.5

>241 TPI 1567-5 —401 —3.25

209 TPI 1577-6 —476 —4.20

113 TPI 1577-7 —495 —4.57

71 TPI 1577-8 —491 —4.39

88 TPI 1567-11 —505 —4.58

75 TPI 1567-12 —525 —4.25

124 TPI 1567-13 —633 —4.53

58 TPI 1567-14 —665 —4.98

81 TPI 1577-9 —557 —4.44

23 TPI 1567-23 —628 —3.52

35 TPI 1567-24 —523 —3.13

122 TPI 1567-18 —629 —4.39

61 TPI 1572-8 —353 —2.63

>283 TPI 1572-15 —515 —2.97

171 TPI 1572-16 —529 —3.16

149 TPI 1572-10 —477 —3.29

>210 TPI 1572-11 —505 —3.68

89 TPI 1572-14 —506 —3.89

103 TPI 1572-17 —506 —3.89

132 TPI 1572-18 —581 —4.95

164 TPI 1572-19 —457 —3.01

>219 TPI 1572-20 —515 —2.7

235 TPI 1572-21 —639 —4.61

69 TPI 1572-22 —647 —4.7

79 TPI 1572-23 —701 —4.39

94¹Molecular weight in grams/mole.²Relative caspase-3 activity in the XIAP derepression assay wascalculated as the ratio of the Vmax in the presence of each compounddivided by the Vmax of the controls containing caspase-3 and XIAP butlacking the compounds.

Following the procedure of Example XI, TPI 1540-14, TPI 1567-11, TPI1567-12, TPI 1567-13, TPI 1567-14, TPI 1577-9 and TPI 1509-7 were testedfor their ability to induce apoptosis in Jurkat cells. The results ofthese tests are shown in FIG. 54. As compared to the DMSO control, eachof the tested compounds proved capable of inducing apoptosis in thetested cell line at micromolar concentrations.

EXAMPLE XXIX Scintillation Proximity Assay

This example describes a scintillation proximity assay. This method usesderepressors of IAP-inhibited caspase modified with a radiolabel, suchas tritium, in a scintillation proximity assay (SPA). In a homogeneousassay, copper chelate (His-Tag) YSi SPA™ Scintillation Beads (availablefrom Amersham-Pharmacia) are mixed with His-BIR2, His-BIR1-2 orHis-BIR1-2-3 and a radiolabeled compound. Unlabeled competing compounds(candidate compounds) are then added at various concentrations in 96well plates which are spun down, pelleting the beads, attachedHis-protein and bound radiolabel. Plates are then read in ascintillation counter. Reduction in bound radiolabel reflectscompetition by unlabeled candidate compounds. Candidate compounds thatcompetitively displace one or more labeled derepressors are identifiedas ligands and potential derepressors of IAP inhibited caspase. SeeAlderton, W. K. and P. N. Lowe, 1999, “Scintillation Proximity Assay toMeasure Nitroargine and Tetrahydrobioperin Binding to Heme Domain ofNeuronal Nitric Oxide Synthase,” Methods in Enzymol., 301:114-125.

EXAMPLE XXX Scintillation Proximity Assay

This assay utilizes Ni-NTA Hi Sorb Plates™ from Qiagen to identifycandidate compounds that are derepressors of IAP inhibited caspase. Thisis a non-homogenous assay with several washing steps. One or more ofHis-BIR2, His-BIR1-2 or His-BIR1-2-3 are bound to a plate. Abiotin-labeled compound, a fluorophore-labeled compound or aradiolabeled compound is then bound to the protein in the presence ofvarying concentrations of competing unlabeled compound (candidatecompound). Following at least one wash step, bound labeled compound ismeasured. For example, where the labeled compound is biotinylated, theread-out is via alkaline or horseradish peroxidase conjugatedstreptavidine (both available from Amersham-Pharmacia), which yields anabsorbance in the visible range, which can be measured with aspectrophotometer. Where the labeled compound is radiolabeled,radioactive read-out is obtained with a scintillation counter. Where thelabeled compound is fluorescently labeled, fluorescence read-out isobtained with a fluorescence spectrophotometer. A decrease in theread-out in the presence of competing unlabeled compound (candidatecompound) reflects competition by the unlabeled compound. A candidatecompound that competitively displaced labeled compound is identified asa ligand and as a potential derepressor of IAP inhibited caspase.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. An isolated agent comprising one of the compounds identified hereinas TPI 1577-1, TPI 1577-2, TPI 1577-3, TPI 1567-5, TPI 1577-6, TPI1577-7, TPI 1577-8, TPI 1567-11, TPI 1567-12, TPI 1567-13, TPI 1567-14,TPI 1577-9, TPI 1567-23, TPI 1567-24, TPI 1567-18, TPI 1572-8, TPI1572-15, TPI 1572-16, TPI 1572-10, TPI 1572-11, TPI 1572-14, TPI1572-17, TPI 1572-18, TPI 1572-19, TPI 1572-20, TPI 1572-21, TPI 1572-22or TPI 1572-23.
 2. A composition comprising an isolated agent of claim 1in admixture with a diluent.
 3. A pharmaceutical composition comprisingthe agent of claim 1 and a pharmaceutically acceptable carrier.
 4. Acomplex comprising an IAP bound to an agent selected from the groupconsisting of a one of the compounds set forth in claim
 1. 5. Thecomplex of claim 4, wherein said IAP is selected from the groupconsisting of XIAP, c-IAP-1, c-IAP-2, NIAP, BRUCE (Appollon), ML-IAP,ILP2, DIAP-1, DIAP-2 and survivin.
 6. A method of derepressing anIAP-inhibited caspase, comprising contacting an IAP-inhibited caspasewith an effective amount of an agent to derepress an IAP-inhibitedcaspase, said agent being selected from the group of agents set forth inclaim
 1. 7. The method of claim 6, wherein said IAP is selected from thegroup consisting of XIAP, c-IAP-1, c-IAP-2, NIAP, BRUCE (Appollon),ML-IAP, ILP2, DIAP-1, DIAP-2 and survivin.
 8. The method of claim 6,wherein said caspase is selected from the group consisting of caspase-3,caspase-7 and caspase-9 and the drosophila caspases DCP-1, DRICE andDRONC.
 9. The method of claim 6, wherein said contacting is performed invitro.
 10. The method of claim 6, wherein said contacting occurs in acell.
 11. A method of promoting apoptosis in a cell, comprisingcontacting a cell with an effective amount of an agent to derepress anIAP-inhibited caspase, said agent having being selected from the groupof agents set forth in claim
 1. 12. The method of claim 11, wherein saidcell is a eukaryotic cell.
 13. The method of claim 11, wherein said IAPis selected from the group consisting of XIAP, c-IAP-1, c-IAP-2, NIAP,BRUCE (Appollon), ML-IAP, ILP2, DIAP-1, DIAP-2 and survivin.
 14. Themethod of claim 11, wherein said caspase is selected from the groupconsisting of caspase-3, caspase-7 and caspase-9 and the drosophilacaspases DCP-1, DRICE and DRONC.
 15. A method of reducing the severityof a pathologic condition in an individual, comprising administering toan individual having a pathologic condition characterized by apathologically reduced level of apoptosis, an effective amount of anagent to derepress an IAP-inhibited caspase, said agent being selectedfrom the group of agents set forth in claim
 1. 16. The method of claim15, wherein said pathologic condition is cancer.
 17. The method of claim15, wherein said pathologic condition is selected from the groupconsisting of psoriasis, hyperplasia, an autoimmune disease andrestenosis.
 18. The method of claim 15, wherein said IAP is selectedfrom the group consisting of XIAP, c-IAP-1, c-IAP-2, NIAP, BRUCE(Appollon), ML-IAP, ILP2, DIAP-1, DIAP-2 and survivin.
 19. The method ofclaim 15, wherein said caspase is selected from the group consisting ofcaspase-3, caspase-7 and caspase-9 and the drosophila caspases DCP-1,DRICE and DRONC.
 20. The method of claim 15, further comprisingadministering a second therapeutic agent. 21-64. (canceled)